https://wiki.oroboros.at/api.php?action=feedcontributions&user=Hiller+Elisabeth&feedformat=atomBioblast - User contributions [en]2024-03-28T10:35:55ZUser contributionsMediaWiki 1.36.1https://wiki.oroboros.at/index.php?title=MiPNet02.05_DatLab2_O2Kinetics&diff=149235MiPNet02.05 DatLab2 O2Kinetics2018-01-11T09:29:18Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=http://wiki.oroboros.at/index.php/O2k-Manual|O2k-Manual]] DatLab 2. Analysis of oxygen kinetics. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/e/e2/MiPNet02.05_DatLab2_O2Kinetics.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet02.05_DatLab2_O2Kinetics.pdf Versions]<br />
|authors=Oroboros <br />
|year=1997-01<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Gnaiger E, Lassnig B (1997) DatLab 2. Analysis of oxygen kinetics. Mitochondr Physiol Network 02.05.''' <br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]<br />
|keywords=DatLab2<br />
|mipnetlab=AT_Innsbruck_Oroboros <br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|topics=Oxygen kinetics<br />
|instruments=Oxygraph-2k, O2k-Manual<br />
|additional=DatLab2<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet02.07_Datlab2_Manual&diff=149234MiPNet02.07 Datlab2 Manual2018-01-11T09:28:56Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=http://wiki.oroboros.at/index.php/O2k-Manual|O2k-Manual]] DatLab 2 Analysis. High resolution of data in the lab. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/8/83/MiPNet02.07_Datlab2_Manual.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet02.07_Datlab2_Manual.pdf Versions]<br />
|authors=Oroboros<br />
|year=1997<br />
|journal=Mitochondr Physiol Network<br />
|abstract= '''Gnaiger E, Reck M (1997) DatLab 2 Analysis. High resolution of data in the lab. Mitochondr Physiol Network 02.07: 1-72.''' <br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue ]]<br />
|keywords=Archive, DatLab2<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=O2k-Manual<br />
|additional=DatLab2<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet03.02_Chemicals-Media&diff=149233MiPNet03.02 Chemicals-Media2018-01-11T09:28:34Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] Selected media and chemicals for respirometry with mitochondrial preparations.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/3/3c/MiPNet03.02_Chemicals-Media.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet03.02_Chemicals-Media.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-08-30<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fontana-Ayoub M, Fasching M, Gnaiger E (2016) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(18):1-10.'''<br />
<br />
Different media for tissue preparation and respiration are used in investigations of mitochondrial function. Initial decisions on the composition of media and chemicals are decisive for long-term studies and crucial for comparability of results. As a guideline, we summarize an update of our experience with media and chemicals for high-resolution respirometry with isolated mitochondria, permeabilized cells, muscle fibres and tissue homogenates. Whereas optimization is necessary for specific experimental protocols, standardization will improve the comparability of results obtained in different laboratories. Efforts towards standardization are important for the advancement of mitochondrial physiology.<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT Innsbruck Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=O2k-Protocol<br />
|additional=O2k-chemicals and media<br />
}}<br />
== Supplementary information ==<br />
::::» [[MiPNet09.12 O2k-Titrations]]; [[MitoPedia]]<br />
::::» [[Cytochrome c control factor]]<br />
::::» For calculations, see Excel file: [http://www.oroboros.at/?Protocols_titrations O2k-Titrations.xls]<br />
::::» For pH adjustment of BIOPS solution, note the temperature dependence:<br />
:::::::* at 0 °C pH = 7.1<br />
:::::::* at 23 °C pH = 6.75<br />
<br />
== History ==<br />
<br />
::::* Malate was listed for a final concentration of 2 mM until 2013. During this year, test experiments with mitochondria from various tissues and species and with different mt-preparations (isolated mitochondria, permeabilized fibres, tissue homogenate) revealed an inhibitory effect of 2 mM malate on CII linked respiration (S,Rot). The inhibitory effect is less at 0.5 mM malate, and 0.5 mM malate may be saturating for CI-linked respiration. Evaluation is required for the optimum malate concentrations in SUIT protocols with sequential CI, CI<small>&</small>II and CII substrate states. <br />
<br />
::::* [[Carbonyl cyanide m-chlorophenyl hydrazone|From FCCP to CCCP]]<br />
<br />
::::* Up to 2006, rotenone was added at a high final concentration (0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggested to use a stock solution at lower concentration (0.1 mM), to reduce the problem of rotenone retention. The 2006 edition listed a 0.2 mM stock solution, but the amount added to 10 ml ethanol was given for a 0.15 mM stock solution. Our recent rotenone titrations with permeabilized muscle fibers, however, confirmed that the originally proposed higher rotenone concentration is actually required for full inhibition.</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet06.01_O2k-Overview&diff=149232MiPNet06.01 O2k-Overview2018-01-11T09:27:55Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:Logo OROBOROS INSTRUMENTS.jpg|right|60px|link=http://www.oroboros.at|Oroboros ]]Gnaiger E (2001) The Oxygraph for high-resolution respirometry. Mitochondr Physiol Network 06.01.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/7/76/MiPNet06.01_O2k-Overview.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet06.01_O2k-Overview.pdf Versions]<br />
|authors=Oroboros <br />
|year=2001<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Summary:''' The Oroboros O2k provides the instrumental basis for high-resolution respirometry. Compared to any of its competitors, the Oroboros O2k is a high-performance instrument, and high-resolution is distinguished from conventional approaches by a combination of unique features and specifications. These set a new standard in bioenergetics, mitochondrial physiology, clinical research and diagnosis of mitochondrial pathologies. <br />
|keywords=high-resolution respirometry<br />
|mipnetlab=AT Innsbruck Oroboros ,<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|instruments=Oxygraph-2k<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet06.03_POS-calibration-SOP&diff=149231MiPNet06.03 POS-calibration-SOP2018-01-11T09:27:38Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k Quality Control 1: Polarographic oxygen sensors and accuracy of calibration.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/7/77/MiPNet06.03_POS-Calibration-SOP.pdf |Bioblast pdf]] » [http://www.bioblast.at/index.php/File:MiPNet06.03_POS-Calibration-SOP.pdf Versions]|authors=Oroboros<br />
|year=2018-01-07<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Gnaiger E (2018) O2k Quality Control 1: Polarographic oxygen sensors and accuracy of calibration. Mitochondr Physiol Network 06.03(17):1-20.'''<br />
[[Image:MitoPedia.jpg|left|60px|link=http://www.bioblast.at/index.php/MitoPedia: DatLab|MitoPedia: DatLab]] [[O2 calibration - DatLab]]<br />
{{MiPNet pdf page linking to MitoPedia}}<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-SOP<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet06.05_Test_Experiments_on_O2k-Specifications&diff=149230MiPNet06.05 Test Experiments on O2k-Specifications2018-01-11T09:27:11Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:Logo OROBOROS INSTRUMENTS.jpg|right|60px|link=OROBOROS INSTRUMENTS|OROBOROS]]Gnaiger E (2010) Test experiments on specifications of the Oroboros Oxygraph. Mitochondr Physiol Network 6.5. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/94/MiPNet06.05_O2k-Specifications.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet06.05_O2k-Specifications.pdf Versions]<br />
|authors=Oroboros<br />
|year=2010<br />
|journal=Mitochondr Physiol Network<br />
|abstract=Summary: The Oroboros O2k with DatLab software is world-wide the only instrument on the market, which allows routine measurements to be made with specifications summarized under the term “high-resolution respirometry”: The limit of detection of O<sub>2</sub> flux is as low as 0.5 pmol.s<sup>-1</sup>.cm<sup>-3</sup>. Signal noise at zero oxygen concentration is <0.05 μM O<sub>2</sub>. Oxygen backdiffusion at zero oxygen is <3 pmol.s<sup>-1</sup>.cm<sup>-3</sup>, and oxygen consumption at air saturation and standard barometric pressure (100 kPa) was 2.7 +/- 0.9 SD in 114 test runs at 37 °C. These highly standardized instrumental background fluxes are a linear function of oxygen concentration, which is used for routine background correction of oxygen flux. Typical exponential time constants of the oxygen sensors are <4 s, used for dynamic corrections in kinetic studies. Results of an extensive experimental test program are presented as the basis of Oroboros O2k specifications, which distinguish high-resolution respirometry from any conventional oxygraph system. <br />
|mipnetlab=AT Innsbruck Oroboros<br />
}}<br />
[[File:Flyer backside .png|right|450px]]<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=Oxygraph-2k<br />
}}<br />
<br />
== O2k high-resolution respirometry ==<br />
:::: The Oroboros O2k with DatLab software is world-wide the only instrument (sole source) which allows routine measurements to be made with specifications summarized under the term [[O2k high-resolution respirometry |'''high-resolution respirometry (HRR)''']].<br />
<br />
:::: Continuous control of the Oroboros O2k specifications are a fundamental component of [[Quality Assurance]] and distinguish high-resolution respirometry from any conventional oxygraph system and from less quantitative approaches.<br />
::::» [[O2k specifications]]<br />
<br />
<br />
<br />
== Oroboros O2k: technical specifications ==<br />
<br />
<br />
<br />
=== General specifications ===<br />
<br />
:::* Dimensions - O2k-Main Unit: L 45 cm, W 31 cm, H 25 cm; 13.45 kg without packing box or Peli Case.<br />
<br />
:::* Chambers: chemically inert and minimum oxygen diffusion:<br />
<br />
:::::: - Two Duran glass chambers with 2 ml (1.5 to 3.5 ml) volume; 16 mm inner diameter.<br />
<br />
:::::: - PVDF or PEEK stoppers with Viton O-rings.<br />
<br />
:::::: - Electromagnetic stirrers with variable speed (100 to 900 rpm).<br />
<br />
:::::: - PVDF- or PEEK-coated stirrer bars (6 mm diameter).<br />
<br />
:::::: - Polarographic oxygen sensors (OroboPOS) sealed with butyl rubber seal tips.<br />
<br />
:::* Built-in electronically regulated Peltier thermostat:<br />
<br />
:::::: - Temperature range:<br />
::::::{| class="wikitable" <br />
|-<br />
| At room temperature <br />
| 4 °C to 47 °C <br />
|-<br />
| At lower ambient temperature<br />
| 2 °C<br />
|}<br />
<br />
:::::: - Temperature stability:<br />
::::::{| class="wikitable" <br />
|-<br />
| Temperature stability <br />
| ±0.002 °C over 90 min.<br />
|}<br />
<br />
:::::: - Temperature range:<br />
::::::{| class="wikitable" <br />
|-<br />
| in 15 min<br />
| 20 °C to 30 °C <br />
|-<br />
| in 20 min<br />
| 30 °C to 20 °C<br />
|}<br />
<br />
:::* Data output through USB:<br />
<br />
:::::: - Two oxygen signals.<br />
<br />
:::::: - Pressure transducer, for absolute barometric pressure signal, resolution 0.1 kPa.<br />
<br />
:::::: - Thermostat temperature signal, resolution 0.001 °C.<br />
<br />
:::::: - O2k-MultiSensor: two additional amperometric signals (O2k-fluorescence; NO sensors), and two additional potentiometric signals pX: for TPP or pH).<br />
<br />
:::* DatLab software:<br />
<br />
:::::: - Simultaneous display of O2 concentration and O2 flux (negative slope or time derivative), together with any selected O2k-MultiSensor channels (raw or calibrated signals and corresponding time derivatives). <br />
<br />
:::::: - Barometric pressure for O2 calibration; temperature stability control.<br />
<br />
:::::: - Automatic calibrations: O2 and O2k-MultiSensor channels.<br />
<br />
:::::: - DL-Protocols: real-time guide through instrumental quality control tests and advanced diagnostic substrate-uncoupler-inhibitor titration (SUIT) protocols within a single assay for exploring mitochondrial function.<br />
<br />
<br />
<br />
<br />
=== Specifications for high-resolution respirometry === <br />
<br />
:::* Oxygen signal:<br />
<br />
:::::: - Noise at zero O<sub>2</sub>: <±0.02 µM (SD, 100 data points recorded at 0.2 s intervals at 37 °C) without smoothing (±0.005 µM typical)<br />
<br />
:::::: - Noise at zero O<sub>2</sub>: <±0.002 kPa (SD, 100 data points recorded at 0.2 s intervals) without smoothing (±0.0005 kPa typical).<br />
<br />
:::::: - Noise at air saturation: <±0.1 µM O<sub>2</sub> (SD, 100 data points recorded 0.2 s intervals at 37 °C) without smoothing, at 180 µM O<sub>2</sub> (±0.05 µM typical).<br />
<br />
:::::: - Noise at air saturation: <±0.01 kPa (SD, 100 data points recorded 0.2 s intervals) without smoothing, at partial oxygen pressure of 20 kPa (±0.005 kPa typical).<br />
<br />
:::::: - O<sub>2</sub> range of linearity: 0 to 1000 µM.<br />
<br />
:::::: - Time constant: <4 s at 37 °C (<3 s typical).<br />
<br />
<br />
:::* Oxygen flux ''J<sub>O2,V</sub>'' [pmol.s<sup>-1</sup>.ml<sup>-1</sup>]:<br />
<br />
:::::: - Limit of detection: 0.5 at steady-state over 5 min.<br />
<br />
:::::: - Sensitivity (normoxic): <1 at steady-state over 5 min at 20-40 °C.<br />
<br />
:::::: - Sensitivity (hyperoxic): <3 at steady-state over 5 min at 20-40 °C.<br />
<br />
:::::: - Noise: <0.2 after standard smoothing (120 s).<br />
<br />
:::::: - O<sub>2</sub> range: flux measured up to 500 µM O<sub>2</sub> (permeabilized fibres), and <0.1 µM based on DatLab analysis of oxygen kinetics (mitochondria and cells).<br />
<br />
<br />
:::* Instrumental background for linear correction over the entire oxygen range:<br />
<br />
:::::: - O<sub>2</sub> backdiffusion [pmol.s<sup>-1</sup>.ml<sup>-1</sup>] at 0 µM: <3 at 20-40 °C (2.5 typical).<br />
<br />
:::::: - O<sub>2</sub> backdiffusion [pmol.s<sup>-1</sup>.ml<sup>-1</sup>] at 0 kPa: <3 at 20-40 °C (2.5 typical).<br />
<br />
:::::: - O<sub>2</sub> consumption [pmol.s<sup>-1</sup>.ml<sup>-1</sup>] at 200 µM: <4 at 37 °C (3 typical); <3 at 25 °C (2 typical).<br />
<br />
:::::: - O<sub>2</sub> consumption [pmol.s<sup>-1</sup>.ml<sup>-1</sup>] at 20 kPa: <4 at 37 °C (3 typical); <3 at 25 °C (2 typical).<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet06.06_Chemical_O2_background&diff=149229MiPNet06.06 Chemical O2 background2018-01-11T09:26:47Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Oxygraph assay of cytochrome ''c'' oxidase activity: chemical background correction.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/1/1b/MiPNet06.06_ChemicalO2Background.pdf |Bioblast pdf]] » [http://www.bioblast.at/index.php/File:MiPNet06.06_ChemicalO2Background.pdf Versions]<br />
|authors=Oroboros<br />
|year=2015-04-27<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Kuznetsov AV, Gnaiger E (2010) Oxygraph assay of cytochrome ''c'' oxidase activity: chemical background correction. Mitochondr Physiol Network 06.06(07):1-4.''' <br />
<br />
Autoxidation of reduced compounds (such as ascorbate, TMPD, cytochrome c) causes a chemical background oxygen flux, which is a function of oxygen concentration and has to be subtracted fom total oxygen flux. The new DatLab software provides on-line correction for instrumental and chemical background.<br />
<br />
Oxygraphic determination of cytochrome c oxidase activity in the presence of TMPD, ascorbate and cytochrome c requires consideration of chemical background oxygen consumption. Several compounds are readily oxidized by molecular oxygen when dissolved in water. This leads to a significant chemical oxygen consumption in the absence of any respiring biological sample. The rate of this autoxidation represents a chemical background which strongly depends on experimental conditions, such as temperature, chemical composition, pH and oxygen concentration. Moreover, the rate of autoxidation may be significantly catalyzed by metal traces and metal-containing proteins (e.g. cytochrome c). Therefore, high-resolution respirometric measurement of oxygen flux ultimately depends on chemical background correction analyzed under experimental conditions over the entire oxygen range.<br />
<br />
After sequential titration of ascorbate (added first) and TMPD (with or without added cytochrome c), the total oxygen flux increases due to (i) COX activity and (ii) autooxidation of ascrobate and TMPD. Correction for autooxidation is a routine procedure in high-resolution respirometry, but it requires precautions. We recommend, therefore, evaluation of chemical background effects by application of inhibitors of cytochrome c oxidase (cyanide, azide) subsequent to recording of flux with ascorbate and TMPD. After full inhibitioin of COX, a small non-COX component of biological oxidations and the large effect of autooxidation are recorded over a range of further declining oxygen concentration. After re-oxygenation, inhibited oxygen flux is recorded at oxygen levels above and within the oxygen concentration pertaining to the COX activity measurment. This provides a complete internal calibration of the chemical background, as a function of oxygen concentration.<br />
:>> Product: O2k-Catalogue: [[O2k-MultiSensor]], [[O2k-Core]], [[O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|preparations=Enzyme, Oxidase;biochemical oxidation<br />
|enzymes=Complex IV;cytochrome c oxidase<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-SOP<br />
}}<br />
<table><br />
<tr><br />
<td>[[Image:MiPNet06.06_Fig1.jpg|center|400px|thumb]]</td><br />
<td>[[Image:MiPNet06.06_Fig2.jpg|center|400px|thumb]]</td><br />
</tr><br />
<tr><br />
<td><span style="color:grey">Fig. 1. Continuous records of chemical background flux with 0.5 mM TMPD, 2 mM ascorbate,<br> and various cytochrome c concentrations (μM, indicated as numbers at each curve), plotted as a <br> function of oxygen concentration. Measured at 30 °C in mitochondrial medium R05 with 280 IU/ml <br> catalase. The linear sections of the curves were used for background corrections at high oxygen >50 μM.</span></td><br />
<td align:right><span style="color:grey; align:right">Fig. 2. Parameters of chemical background oxygen flux with TMPD+ascorbate as a <br> function of cytochrome c concentration (Cyt c [μM). ao’ intercept and bo’ slope of <br>linear part of oxygen dependence, from Fig.1 </span></td><br />
</tr><br />
</table><br />
'''References'''<br />
<br />
[[Renner_2002_Mol_Biol_Rep| Renner K, Kofler R, Gnaiger E (2002) Mitochondrial function in glucocorticoid triggered T-ALL cells with transgenic Bcl-2 expression. Mol Biol Rep 29: 97-101.]]<br />
<br />
<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet08.09_CellRespiration&diff=149218MiPNet08.09 CellRespiration2018-01-11T09:06:38Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ET-pathway, ROX.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/d/da/MiPNet08.09_CellRespiration.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet08.09_CellRespiration.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-08-11<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Doerrier C, Gnaiger E (2003-2016) High-resolution respirometry and coupling control protocol with intact cells: ROUTINE, LEAK, ET-pathway, ROX. Mitochondr Physiol Network 08.09(11):1-8.'''<br />
<br />
An experiment on respiration of [[intact cells]] is reported from an O2k-Workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k-Chambers operated in parallel. Cellular ROUTINE respiration, ''J''<sub>R</sub>, resulted in volume-specific oxygen consumption of 20 pmol·s<sup>-1</sup>·ml<sup>-1</sup>. Oxygen concentration changed by merely 6.4 and 6.5 µM in the two O2k-Chambers over a period of 5 min (<1% air saturation per minute). Inhibition by oligomycin (''J<sub>L</sub>''), and rotenone (residual oxygen consumption, ''J''<sub>ROX</sub>; after uncoupling) reduced respiration to 5 and 1 pmol·s<sup>-1</sup>·ml<sup>-1</sup>, while inducing the noncoupled state by the uncoupler FCCP revealed the capacity of the Electron transfer-pathway (ET-pathway) at ''J<sub>E</sub>'' of 50 pmol·s<sup>-1</sup>·ml<sup>-1</sup>. The ROUTINE control ratio, ''R/E'', was 0.4 (uncoupling control ratio, UCR=''E/R''=2.5), and the LEAK control ratio, ''L/E'', was 0.1 (''E/L''=12.0). This indicates tight coupling of OXPHOS, and a large ET-pathway excess capacity over ROUTINE respiration. The net ROUTINE control ratio, net''R''=(''R-L'')/''E'' was 0.30, indicating that 30% of ET-pathway capacity was activated for ATP production.<br />
<br />
Automatic correction for instrumental background amounted to 13% for ROUTINE respiration, but to >50% and 180% for ''J<sub>L</sub>'' and ''J''<sub>ROX</sub>, respectively, illustrating the importance of real-time correction. The experiment illustrates the sensitivity and resproducibility of high-resolution respirometry with the OROBOROS O2k. Calibrations and routine corrections provide the basis of the high accuracy required for mitochondrial respiratory physiology. Real-time analyses were performed, combining high-resolution with instant diagnostic information. In this update graphs are presented illustrating some features of DatLab.<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Human<br />
|tissues=Blood cells, Lymphocyte<br />
|preparations=Intact cells<br />
|couplingstates=LEAK, ROUTINE, ET<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-Demo, O2k-Core, 1ce;2ceOmy;3ceU-<br />
}}<br />
'''»''' [[MitoPedia: Respiratory states]]<br />
[[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:L.jpg|link=LEAK respiration|LEAK]] [[Image:E.jpg|link=ET-capacity|ET-capacity]] [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]]<br />
[[Image:MiPNet0809_FCR.jpg|left|800px|]] Flux control ratios (''FCR'') normalized to state ET-pathway (''E'') in chambers A and B (superimposed). ROUTINE respiration in these cells is regulated at 0.4 of ET-capacity (''R/E''). Oligomycin (Omy) inhibits respiration to 0.1 ET-capacity (''L/E'').<br />
<br />
<br />
== Coupling control protocol ==<br />
[[File:1ce;2ceOmy;3ceU-.jpg|right|300px|link=1ce;2ceOmy;3ceU-]]<br />
****: ceCCP: RLE [[1ce;2ceOmy;3ceU-]]<br />
::::» Instructions for using templates for data evaluation are given in MiPNet08.09 (see above) and [[MiPNet10.04 CellRespiration]].<br />
<br />
'''ceCCP states'''<br />
{| class="wikitable" border="1"<br />
|-! Step<br />
! Respiratory state<br />
! Mark names<br />
! Explanations<br />
|-<br />
| ''R''<br />
| 1ce<br />
| [[ROUTINE]]<br />
|-<br />
| ''L''<br />
| 2ceOmy<br />
| [[Oligomycin]]<br />
|-<br />
| ''E''<br />
| 3ceU<br />
| [[Uncoupler]] titration<br />
|-<br />
| ROX<br />
| 4ceRotAma<br />
| [[Rotenone]], [[Antimycin A]], residual oxygen consumption<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== DatLab-Analysis templates and DatLab Demo Files ==<br />
<br />
=== DatLab 7 ===<br />
<br />
::Calibration-File: [[Media:MiPNet08.09_CellRespiration_Calib.DLD|MiPNet08.09_CellRespiration_Calib.DLD]]<br />
<br />
::Demo-File: [[Media:MIPNET08.09 CellRespiration.DLD|MiPNet08.09_CellRespiration.DLD]]<br />
<br />
::DatLab-Analysis template: [[Media:SUIT MiPNet08.09 CellRespiration.xlsx|CCP02.xlsx]]<br />
<br />
=== DatLab 6 ===<br />
<br />
::Calibration-File: [[Media:MIPNET08.09 2003-03-29 P1-01 CALIB.DLD|MIPNET08.09_2003-03-29_P1-01_CALIB.DLD]]<br />
<br />
::Demo-File: [[Media:MIPNET08.09 2003-03-29 P1-02 CELLS.DLD|MIPNET08.09_2003-03-29_P1-02_CELLS.DLD]]<br />
<br />
::Background Excel demo and template: [[Media:MiPNet08.09 O2k-Background Cells DL6.xlsx|MiPNet08.09_O2k-Background_Cells.xlsx]]<br />
<br />
::DatLab-Analysis template: [[Media:MiPNet08.09 O2k-Analysis Cells DL6.xlsx|MiPNet08.09_O2k-Analysis_Cells.xlsx]]<br />
<br />
=== DatLab 5 ===<br />
<br />
::Calibration-File: [[Media:MIPNET08.09 2003-03-29 P1-01 CALIB.DLD|MIPNET08.09_2003-03-29_P1-01_CALIB.DLD]]<br />
<br />
::Demo-File: [[Media:MIPNET08.09 2003-03-29 P1-02 CELLS.DLD|MIPNET08.09_2003-03-29_P1-02_CELLS.DLD]]<br />
<br />
::Background Excel demo and template: [[Media:O2k-Background Cells 0809 DL5.xlsx|O2k-Background_Cells_0809.xlsx]]<br />
<br />
::DatLab-Analysis template: [[Media:O2k-Analysis Cells 0809 DL5.xlsx|O2k-Analysis_Cells_0809.xlsx]]<br />
<br />
== Further information ==<br />
::::» [[Intact cells]]<br />
::::» [[Coupling control protocol]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet08.13_mt-Isolation-RLM&diff=149216MiPNet08.13 mt-Isolation-RLM2018-01-11T09:06:11Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Laboratory protocol: Isolation of rat liver mitochondria. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/93/MiPNet08.13_MitoIsolation-RLM.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet08.13_MitoIsolation-RLM.pdf Versions]<br />
|authors=Oroboros<br />
|year=2010-08-03<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Lassnig B, Gnaiger E. Laboratory protocol: Isolation of rat liver mitochondria. Mitochondr Physiol Network 08.13.''' <br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|organism=Rat<br />
|tissues=Liver<br />
|preparations=Isolated mitochondria<br />
|instruments=O2k-Protocol<br />
|additional=Mt-Preparation<br />
}}<br />
== Further information ==<br />
<br />
:>> [http://bioblast.at/images/3/3c/MiPNet03.02_Chemicals-Media.pdf Chemicals and Media]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet08.15_Complex-I&diff=149215MiPNet08.15 Complex-I2018-01-11T09:05:44Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Laboratory protocol: Complex I (NADH:Ubiquinone Oxidoreductase, EC 1.6.5.3). Mitochondrial membrane enzyme. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/40/MiPNet08.15_Complex-I.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet08.15_Complex-I.pdf Versions]<br />
|authors=Oroboros<br />
|year=2010-08-23<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Kuznetsov AV, Gnaiger E. Laboratory protocol: Complex I (NADH:Ubiquinone Oxidoreductase, EC 1.6.5.3). Mitochondrial membrane enzyme. Mitochondr Physiol Network 08.15.'''<br />
<br />
Complex I (CI) is the segment of the electron transport system (integral enzyme of the inner mitochondrial membrane) responsible for electron transfer from NADH to ubiquinone. CI is sensitive to different pathologies, particularly to oxidative stress, which is involved in ischemia-reperfusion injury, anoxia/ reoxygenation, aging, etc (Kuznetsov et al 2004; Rouslin & Millard 1981; Rouslin & Ranganathan, 1983; Rouslin, 1983). For the assessment of CI activity, among the ubiquinone isoprenologs, it is most convenient to use ubiquinone-1 (CoQ1) as electron acceptor, because of its relative water solubility. Importantly, CoQ1 yields one of the lowest rotenone insensitive rates and a high enzymatic rate. It is, therefore, the best electron acceptors for the CI assay.<br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|preparations=Enzyme<br />
|enzymes=Complex I<br />
|instruments=O2k-Protocol<br />
|additional=Mt- and marker-enzymes<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet08.16_pH_calibration&diff=149213MiPNet08.16 pH calibration2018-01-11T09:04:42Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Laboratory protocol: pH measurement and temperature dependence of pH.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/b/be/MiPNet08.16_pH-Calibration.pdf |Bioblast pdf]] » [[Media:MiPNet08.16 pH-Calibration.pdf|Versions]]<br />
|authors=Oroboros<br />
|year=2014-03-06<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Gnaiger E (2014) Laboratory protocol: pH measurement and temperature dependence of pH. Mitochondr Physiol Network 08.16(07):1-4.''' <br />
<br />
pH of blood and intracellular compartments is tightly regulated. Blood pH at 37 °C is 7.4. However, when temperature is lowered, the pH of blood and of intracellular buffer systems increases, which has been reported as early as 1927 but was ignored for the following 30 years. The phenomenon of a change in blood pH with temperature of about -0.016 U/°C was rediscovered by comparative physiologists in studies of "cold blooded" vertebrates (turtles, fish), and is was recognized soon that the same pH/temperature relation applies to "warm blooded" mammals (rat, human). The physicochemical basis of pH/temperature relations and the consequences for acid-base balance and protein function were primarily analyzed by Rahn and Reeves (1979). Besides the importance for physiological and biochemical systems, the temperature dependence of pH of buffer systems has experimental significance for the measurement of pH at different temperatures, and for the choice of buffer systems when designing experiments at various temperatures.<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|keywords=HRR-MultiSensor<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|topics=pH, Temperature<br />
|instruments=pH, O2k-Protocol<br />
|additional=O2k-SOP,<br />
}}<br />
[[Image:MiPNet08.16.jpg|left|300px|thumb|Figure 1. Principle of a two-point calibration of a pH electrode system, at pH 6.00 and 8.00. (a) Signal before calibration (dotted line); (b) Calibration at pH 6 at electronic zero (0 mV), using the zero adjustment (+; additive as shown by the downwards arrows of equal length); (c) calibration of gain setting (slope) at pH 8, observing the pH of 2.0 units (x; multiplicative or proportional, as shown by the constant position of the 0 mV signal); (d) The full line of correspondence is obtained by shifting the entire curve to the correct absolute pH value (+, additive).]] <br />
<br />
<br />
<br />
== Further information ==<br />
<br />
:» [[O2k-pH ISE-Module]]<br />
<br />
:» In [[DatLab]], the two-point calibration calculations are performed automatically (equations in [[MiPNet12.08]]).<br />
<br />
:» Template: [[Media:PH-Calibration-List.xls|pH-Calibration-List.xls]]<br />
<br />
:» [O2k-Protocols| Excel Templates and DatLab-Demo Files]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet08.18_LactateDehydrogenase&diff=149211MiPNet08.18 LactateDehydrogenase2018-01-11T09:03:51Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols contents]]Laboratory protocol: Lactate dehydrogenase. Cytosolic marker enzyme. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/47/MiPNet08.18_LDH.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet08.18_LDH.pdf Versions] <br />
|authors=Oroboros<br />
|year=2010-08-06<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Kuznetsov AV, Gnaiger E. Laboratory protocol: Lactate dehydrogenase. Cytosolic marker enzyme. Mitochondr Physiol Network 08.18.''' <br />
<br />
Lactate dehydrogenase (EC 1.1.1.27) is an enzyme, which catalyzes the last step in glycolysis. LDH is a soluble enzyme and localized in the cytosol (cytoplasm). LDH, therefore, is used as a quantitative marker enzyme for the intact cell, its activity providing information on cellular glycolytic capacity (Renner et al, 2003). Measurement of LDH release (leakage) is an important and frequently applied test for cellular membrane permeabilization (rupture) and severe irreversible cell damage. LDH leakage normally correlates well with CK release and the trypan blue viability test.<br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]<br />
|keywords=Enzymes<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|preparations=Enzyme<br />
|enzymes=Marker enzyme<br />
|instruments=O2k-Protocol<br />
|additional=Mt- and marker-enzymes<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet09.01_O2k-ParadigmShift&diff=149198MiPNet09.01 O2k-ParadigmShift2018-01-11T08:56:14Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=Gnaiger E, Gradl P (2007) Oxygraph-2k: Paradigm shift and features of high-resolution respirometry. Mitochondr Physiol Network 09.01. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/9c/MiPNet09.01_O2k-ParadigmShift.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet09.01_O2k-ParadigmShift.pdf Versions] <br />
|authors=Oroboros<br />
|year=2007<br />
|journal=Mitochondr Physiol Network<br />
|abstract=Modern trends in mitochondrial physiology and mitochondrial respiratory pathology set advanced standards, and present new requirements with respect to high-resolution respirometry of isolated mitochondria, cultured cells, tissue preparations and human biopsies. For more than ten years, the Oroboros O2k is being referred to as the unique instrumental system for high-resolution respirometry. The new Oroboros O2k continues this well appreciated tradition, and extends the experimental options on the basis of chamber design, electronics and the task-specific DatLab software. The Oroboros O2k combines the power of a user-friendly scientific strategy with the skill of professional hardware and software development. The O2k reinforces the scientific strength of an international network dedicated to apply this unique instrument at its best in fundamental science and biomedical research. <br />
<br />
* Version 1: 2007<br />
* Version 5: 2011-12-03<br />
<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
{{Labeling<br />
|instruments=Oxygraph-2k, TIP2k<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
<br />
<br />
== Further Information ==<br />
<br />
[[File:Glass chamber.jpg|right|thumb|300px|The glass chamber and PVDF stirrer of the Oxygraph-2k.]]<br />
:::<big>'''O2k-Chamber: Paradigm shift in HRR'''</big><br />
<br />
::::*The glass chamber and PVDF stirrer of the Oxygraph-2k.<br />
::::*Oxygraph-2k: Paradigm shift and features of high-resolution respirometry. MiPNet09.01. Including sole source information. Bioblast Link<br />
<br />
<br />
::::Small chamber volumes of 20-50 µl generate problems rather than providing resolution with small amounts of tissue samples, low numbers of cultured cells or few isolated mitochondria. The surface to volume ratio increases with decreasing chamber volume, thus various boundary effects entail larger errors at smaller volume, in particular oxygen diffusion. While the rate of oxygen depletion per unit amount of sample increases linearly with decreasing chamber volume, side effects may increase to a larger degree. Then, sensitivity is lost with decreasing chamber size. We recommend a minimum chamber volume of 1.5-2.0 cm3.<br />
<br />
<br />
<br />
:::'''Paradigm shift from mimimum to optimum chamber volume'''<br />
<br />
::::1. Minimization of chamber volume represents a past paradigm, aming at high rates of oxygen consumption per volume. The advantage appears to be obvious, whereas the drawbacks are frequently overlooked (see below).<br />
::::2. Advancements of electronics, data acquisition and analysis, polarographic oxygen sensor specifications and chamber design made possible a superior approach, allowing for respirometric measurements at high dilution, as reviewed by [https://www.ncbi.nlm.nih.gov/pubmed/11718759?dopt=Abstract Gnaiger E (2001)]. In specifically designed mitochondrial respiration media, respiration is stable at high dilution, multiple substrate/inhibitor titrations are possible without oxygen depletion, and a low-oxygen regime may be chosen to prevent elevation of oxidative stress at air-level oxygen saturation. In contrast, micro-chambers are characterized by a high surface-to-volume ratio which hinders optimum stirring, increases unfavourable surface effects and oxygen-backdiffusion, and poses problems with accurate titrations and dilution effects of the sample. These potential - and mostly hidden - artefacts are avoided in high-resolution respirometry, using glass chambers, titanium stoppers, and avoiding teflon-coated stirrers or perspex (yielding high back-diffusion of oxygen).<br />
<br />
::::Assume you have 0.1 mg mitochondrial protein for a respirometric assay. Approach (1) would lead you to search for a 100 µl volume respirometer, to maintain a classical 1 mg/ml protein concentration. In contrast, high-resolution respirometry allows for dilution of mitochondria to 0.02 mg/ml protein. Dilution of 0.1 mg mitochondrial protein in a 2 ml chamber yields an optimum concentration for multiple substrate/inhibitor titrations and kinetic measurements. The high-resolution approach of the Oroboros Oxygraph-2k offers the unique advantages of a versatile and ready-to-use system for studies in mitochondrial physiology and pathology.<br />
<br />
<br />
::::*[[Gnaiger 2012 MitoPathways References|References]]<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet09.12_O2k-Titrations&diff=149197MiPNet09.12 O2k-Titrations2018-01-11T08:55:39Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] O2k manual titrations: SUIT protocols with mitochondrial preparations.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/e/ef/MiPNet09.12_O2k-Titrations.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet09.12_O2k-Titrations.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-09-30<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Oroboros (2016) O2k manual titrations: SUIT protocols with mitochondrial preparations. Mitochondr Physiol Network 09.12(15): 1-2.''' <br />
<br />
A list of substrates, uncouplers, and inhibitors used in [[SUIT]] protocols in [[high-resolution respirometry]], with information on abbreviations, stock concentrations, final experimental concentrations, volumes titrated into the 2 ml O2k-chamber, and syringes used for manual titrations.<br />
:» [[O2k-Protocols]]<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|topics=ADP, Inhibitor, Substrate, Uncoupler, Amino acid<br />
|instruments=O2k-Protocol<br />
|additional=O2k-chemicals and media<br />
}}<br />
<br />
== Note: Titration of chemicals in respirometry ==<br />
<br />
:::: Overshoots or short interruptions of the flux after the injection of chemicals do not have a meaning in respirometric measurements. A disturbance of the signal is only observed due to the injected chemical and does not represent mitochondrial respiration. With the addition of a chemical solved in ethanol a little bit of oxygen is always introduced, since ethanol has higher oxygen solubility than water.<br />
<br />
<br />
== Further information ==<br />
::::» [[MitoPedia: SUIT]] - Substrate-uncoupler-inhibitor-titration (SUIT) protocols for respirometry<br />
::::» [[MiPNet03.02 Chemicals-Media|MiPNet03.02]] - Chemicals and media for respirometry<br />
::::» [[MiPNet19.14 SOP Hamilton microsyringes |MiPNet19.14]] - SOP for manual O2k-titrations with Hamilton microsyringes.<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet10.04_CellRespiration&diff=149196MiPNet10.04 CellRespiration2018-01-11T08:55:07Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] An experiment with high-resolution respirometry: coupling control in cell respiration.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/a/a6/MiPNet10.04_CellRespiration.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet10.04_CellRespiration.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-08-08<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Doerrier C, Gnaiger E (2005-2016) An experiment with high-resolution respirometry: Coupling control in cell respiration. Mitochondr Physiol Network 10.04(09):1-12.'''<br />
<br />
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with cultured, suspended cells in the Oroboros O2k. The experiment demonstrates manual [[MiPNet09.12 O2k-Titrations |titrations]] of inhibitors, and automatic titrations of an uncoupler using the electronic [[O2k-Catalogue: TIP2k |Titration-Injection microPump TIP2k]]. Application of the DatLab 7 software is shown for instrumental control (O2k and TIP2k), and [[MiPNet19.18E O2 flux analysis |real-time data analysis]]. The following guideline describes the experiment in the form of a laboratory protocol, complementary to the relevant sections of the O2k Manual. The experiments were carried out by participants of an O2k-Workshop on high-resolution respirometry in April 2005 (IOC30; Schröcken, Austria).<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT Innsbruck Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Mouse<br />
|tissues=Blood cells<br />
|preparations=Intact cells<br />
|topics=Coupling efficiency;uncoupling, Uncoupler<br />
|couplingstates=LEAK, ROUTINE, ET<br />
|pathways=ROX<br />
|instruments=Oxygraph-2k, TIP2k, O2k-Protocol<br />
|additional=O2k-Demo, O2k-Core, 1ce;2ceOmy;3ceU-<br />
}}<br />
[[Image:MiPNet10.04.jpg|left|500px|]] Respiration of 32D cells (1·10<sup>6</sup> ml<sup>-1</sup>). Traces for the two chambers of the O2k are superimposed. A: Oxygen flow [pmol O<sub>2</sub>·s<sup>-1</sup>·10<sup>6</sup>]; B: Oxygen concentration [μM] (graph layout “05b Specific flux overlay”). After inhibition of ATP synthase with oligomycin, FCCP was titrated at 0.5 μM steps with the TIP2k. Each titration step is automatically marked by an event (vertical lines). After the aerobic-anoxic transition, the chambers were opened for re-oxygenation, causing a 10-min disturbance of the traces of respiratory flow. The TIP2k needles were removed before opening, and re-inserted after closing the chamber.<br />
<br />
<br />
== Cell coupling control protocol ==<br />
****: ceCCP: [[1ce;2ceOmy;3ceU-]]<br />
<br />
== DatLab-Analysis templates and DatLab Demo Files ==<br />
<br />
=== DatLab 7 ===<br />
<br />
::Calibration-File: [[Media:MIPNET10.04_CellRespiration_Calib.DLD|MiPNet10.04_CellRespiration_Calib.DLD]]<br />
<br />
::Demo-File: [[Media:MiPNet10.04_CellRespiration.DLD|MiPNet10.04_CellRespiration.DLD]]<br />
<br />
::Chemical Background Excel demo and template: [[Media:MiPNet10.04_CellRespiration O2k-ChemBackground.xlsx|MiPNet10.04_CellRespiration O2k-ChemBackground.xlsx]]<br />
<br />
::DatLab-Analysis template: [[Media:SUIT_MiPNet10.04_CellRespiration.xlsx|SUIT_MiPNet10.04_CellRespiration.xlsx]]<br />
<br />
== Further information ==<br />
<br />
::::» [[Gnaiger 2008 POS]]<br />
::::» [[Gnaiger 2014 MitoPathways|References]]<br />
::::» [[Coupling control protocol]]<br />
::::» [[DatLab]]<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet10.05_O2-Concentration-Flux&diff=149195MiPNet10.05 O2-Concentration-Flux2018-01-11T08:54:45Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=Gnaiger E (2005-2010) From oxygen concentration to oxygen flux. Mitochondr Physiol Network 10.05. - ''New edition:'' [[Gnaiger_2012_MitoPathways|Gnaiger 2012 Chapter 1]]. <br />
|info= [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/6c/MiPNet10.05_O2-Concentration-Flux.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet10.05_O2-Concentration-Flux.pdf Versions] <br />
|year=2005<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''O2k-Protocol for Oxygen flux''' <br />
<br />
In a closed oxygraph chamber, the oxygen concentration declines over time as a result of respiratory processes. The time derivative, therefore, is a negative number. Why is then the ‘rate of oxygen consumption’ not expressed as a negative value? Why is the term ‘oxygen flux’ used in this context of chemical reactions? The rationale is based on fundamental concepts of physical chemistry and non-equilibrium thermodynamics.<br />
[[Image:O2k-Protocols.jpg|right|150px|link=http://www.oroboros.at/?o2k-protocols|O2k-Protocols contents]]<br />
[[Image:MiPNet10.05.jpg|centre|500px|thumb]]<br />
<br />
Respiratory oxygen flux: On-line display of oxygen concentration (blue) and oxygen flux (respiration, red). Endogenous respiration of endothelial cells leads to oxygen depletion, followed by reoxygenations (dotted arrows). Cell membrane permeabilization by digitonin causes a decline of respiration to the resting level (without adenylates in the medium, -ANP). ADP titration activates respiration about 2-fold above the endogenous level of oxygen consumption.<br />
<br />
Eye-fitted slopes of oxygen chart recorder traces belong to the past. With [[DatLab|DatLab]], trends are resolved. Accuracy is improved by standard numerical corrections. Graphs and protocols are stored and printed ready for publication.<br />
<br />
<br />
'''Reference'''<br />
<br />
[[Gnaiger_1993_Pure_Appl_Chem| Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl Chem 65: 1983-2002.]]<br />
<br />
<br />
<br />
:>> O2k-Protocols:[[O2k-Protocols| Overall contents]]<br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|keywords=Archive<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
{{Labeling<br />
|instruments=O2k-Protocol<br />
|additional=Archive<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
== Further Information==<br />
:>> [[Gnaiger 2012 MitoPathways References]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet11.04_MitoPathways-CI&diff=149190MiPNet11.04 MitoPathways-CI2018-01-11T08:49:55Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=Gnaiger E (2007-2011) Mitochondrial pathways to Complex I: Respiration with pyruvate, malate and glutamate. Mitochondr Physiol Network 11.04. - ''New edition:'' [[Gnaiger 2014 MitoPathways]].<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/3/31/MiPNet11.04_MitoPathways-CI.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet11.04_MitoPathways-CI.pdf Versions]<br />
|year=2007<br />
|journal=Mitochondr Physiol Network<br />
|abstract=<br />
<br />
::»[[Gnaiger 2014 MitoPathways]]<br />
}}<br />
{{Labeling<br />
|additional=Archive<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet11.05_imt-pti-pce&diff=149189MiPNet11.05 imt-pti-pce2018-01-11T08:49:21Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Isolated mitochondria or permeabilized tissues and cells. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/91/MiPNet11.05_Mitos-PermeabilizedCells.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet11.05_Mitos-PermeabilizedCells.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-04-06<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Gnaiger E (2014) Isolated mitochondria or permeabilized tissues and cells. Mitochondr Physiol Network 11.05(06):1-5.''' » [http://www.bioblast.at/index.php/File:MiPNet11.05_Mitos-PermeabilizedCells.pdf Versions]<br />
<br />
Whereas isolated mitochondria remain one of the gold-standards in studies of bioenergetics and mitochondrial physiology, permeabilized tissues and cells have become an alternative with several advantages. But some disadvantages have to be considered, too, for optimum experimental design and critical evaluation of results.<br />
:>> O2k-Protocols:[[O2k-Protocols|Overall contents]]<br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=mt-Biogenesis;mt-density<br />
|preparations=Permeabilized cells, Permeabilized tissue, Isolated mitochondria<br />
|instruments=O2k-Protocol<br />
|additional=Mt-Preparation<br />
}}<br />
== Further information ==<br />
<br />
:::: We recommend the METTLER TOLEDO microbalance XSE105DU for determination of tissue wet weight (1 to 3 mg wet weight per chamber). We offer this microbalance as part of our systems-approach to high-resolution respirometry.<br />
::::» [[Microbalance-Set]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet11.09_MitoPathways-CII&diff=149188MiPNet11.09 MitoPathways-CII2018-01-11T08:48:25Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=Gnaiger E (2007-2011) Mitochondrial pathways to Complex II, glycerophosphate dehydrogenase and electron transferring flavoprotein. Mitochondr Physiol Network 11.09. - ''New edition:'' [[Gnaiger 2014 MitoPathways]].<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/f/f5/MiPNet11.09_MitoPathways-CII.pdf|Bioblast pdf]]<br />
»[http://www.bioblast.at/index.php/File:MiPNet11.09_MitoPathways-CII.pdf Versions]<br />
|authors=Oroboros<br />
|year=2007<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''[[Complex II]] (CII)''' is the only membrane-bound enzyme in the tricarboxylic acid cycle and is part of the mitochondrial [[Electron transfer-pathway]] (ET-pathway). The flavoprotein [[succinate dehydrogenase]] is the largest polypeptide of CII, located on the matrix face of the inner mitochondrial membrane. Following succinate oxidation, the enzyme transfers electrons directly to the quinone pool. Whereas CI is NADH-linked to the dehydrogenases of the [[tricarboxylic acid cycle]] upstream of [[coenzyme Q]], CII is FADH2–linked downstream with subsequent electron flow to Q. [http://www.bioblast.at/index.php/File:MiPNet11.09_MitoPathways-CII.pdf Versions]<br />
|keywords=Archive<br />
|mipnetlab=[[AT_Innsbruck_Oroboros]]<br />
}}<br />
{{Labeling}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.01_Suppl_T-issue&diff=149187MiPNet12.01 Suppl T-issue2018-01-11T08:47:19Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction: mouse skeletal muscle fibres.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/2d/MiPNet12.01_Suppl_T-issue.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.01 Versions]<br />
|authors=Oroboros<br />
|year=2014-07-20<br />
|journal=Mitochondr Physiol Network<br />
|abstract=[[File:MiPNet12.01 SupplementaryT-issue.jpg|left|500px|Suppl T-issue: MitoPathways at the Q-junction]] '''Oroboros (2014) MitoPathways at the Q-junction: mouse skeletal muscle fibres. Mitochondr Physiol Network 12.01(02): Suppl T-issue.''' » [http://www.bioblast.at/index.php/File:MiPNet12.01 Versions]<br />
<br />
[[High-resolution respirometry]] with a [[SUIT protocol]]<sup>1</sup> for [[OXPHOS]] analysis<sup>2</sup> is presented as supplementary '''''T-issue''''' ([[Oroboros]] T-shirt).<br />
<br />
[[Pyruvate]]&[[glutamate]]&[[malate]] (PGM) were used in combination to induce CI-linked [[LEAK respiration]] in permeabilized mouse skeletal muscle ([[MiPNet12.14 IOC39 |IOC39]]; Fig. O2).<sup>3,4</sup> Saturating [[ADP]] (D; 2.5 mM final concentration) stimulated respiration to the level of [[OXPHOS capacity]] (''P'' state), with a small effect of 10 µM [[cytochrome c]] (''c''), expressed as the [[Cytochrome c control factor |cytochrome ''c'' control factor]] (''FCF<sub>c</sub>''<0.01; indicating integrity of the outer mt-membrane). Without correction for residual oxygen consumption (ROX), the biochemical coupling efficiency, (''P-L'')/''P'', was 0.68 (RCR=3.1). Addition of [[succinate]] (S) stimulated respiration by convergent e-input through the [[Q-junction]]. The corresponding succinate control factor was (CI<small>&</small>II-CI)/CI<small>&</small>II=0.47, i.e. succinate increased respiration by 47%. CI<small>&</small>II-linked OXPHOS capacity was not stimulated further by [[uncoupler]] titration (U). Therefore, the capacity of the [[phosphorylation system]] matched the [[ET-capacity]] (''E'' state). At ''E=P'' the [[Excess E-P capacity |Excess ''E-P'' capacity]] is zero, in striking contrast to human skeletal and cardiac muscle mitochondria.<sup>1,5,6</sup> Inhibition of CI by [[rotenone]] (Rot) inhibited respiration to the level of CII-linked ET-capacity. The corresponding CI-control factor is (CI<small>&</small>II-CII)/CI<small>&</small>II=0.25. CII- was higher than CI-linked respiratory capacity (''E=P''). CI<small>&</small>II-linked respiratory capacity was higher than respiration with any single e-input substrate state, indicating an additive effect at the Q-junction. However, since CI<small>&</small>II < CI + CII, the additive effect was incomplete, which indicates that any electron channelling through [[Respiratory complexes |supercomplexes]] to CIV was incomplete. Addition of [[azide]] (Azd) inhibited respiration to the level of [[residual oxygen consumption]] (ROX). ROX was 0.18 of CI<small>&</small>II-linked ET-capacity.<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration<br />
|organism=Mouse<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
|couplingstates=LEAK, ROUTINE, ET<br />
|pathways=N, S, NS, ROX<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=MitoPathways, O2k-Demo, O2k-Core<br />
}}<br />
<ref> Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810:25-58. [[Pesta 2012 Methods Mol Biol |»Bioblast link]] </ref><br />
<ref> Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck:80 pp. [[Gnaiger 2014 MitoPathways |»Bioblast link]] </ref><br />
<ref> Oroboros IOC39. International course on high-resolution respirometry. Schroecken 13-17 April 2007. Mitochondr Physiol Network 12.14: 1-8. [[MiPNet12.14 IOC39 |»Bioblast link]] - O2k-Demo experiment 2007-04-14 A-03 carried out by [[Lemieux H |Hélène Lemieux]] at [[MiPNet12.14 IOC39 |IOC39]], Schröcken. </ref><br />
<ref> Oroboros (2014) Oxygraph-2k manual titrations: SUIT protocols with mitochondrial preparations. Mitochondr Physiol Network 09.12(11): 1. [[MiPNet09.12 O2k-Titrations |»Bioblast link]] </ref><br />
<ref> Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845. [[Gnaiger 2009 Int J Biochem Cell Biol |»Bioblast link]] </ref><br />
<ref> Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43: 1729–38. [[Lemieux 2011 Int J Biochem Cell Biol |»Bioblast link]] </ref><br />
<br />
== Limitations of the SUIT protocol ==<br />
<br />
=== Maximum OXPHOS and ET-capacity ===<br />
:::: Evaluation of maximum respiratory capacities requires titration of further substrates activating additional [[respiratory complexes]] at the Q-junction ([[Electron-transferring flavoprotein complex |CETF]] and [[glycerophosphate dehydrogenase complex |CGpDH<]]).<br />
<br />
=== Malate concentration ===<br />
:::: The [[malate]] concentration was 2 mM, to saturate C<sub>I</sub>-linked respiration. However, at 2 mM malate, the fumarate concentration is increased to a level which inhibits succinate dehydrogenase. Then CI<small>&</small>II- and CII-linked respiratory capacities are underestimated. A malate concentration of 0.5 mM, which saturates CI-linked respiration and inhibits CII-linked respiration to a lesser extent, represents and improved standard.<br />
::::» [[Talk:Malate |Optimum malate concentration in SUIT protocols]]<br />
<br />
=== ROX correction ===<br />
:::: The fact that ROX was higher in the CI<small>&</small>II substrate state compared to CI-linked LEAK respiration indicates that ROX is partially controlled by the substrate state. Therefore, a single measurement of ROX cannot be applied for correction of total oxygen consumption in the different substrate states. Total respiration, therefore, represents apparent coupling states ''L''´, ''P''´ and ''E''´ (Fig. 1). ROX correction is possible in the present experiment only for CI<small>&</small>II- and CII-linked respiration. [[Azide]] inhibits not only CIV but other heme-based oxidases and peroxidases, and therefore may interfere with ROX beyond blocking respiratory electron transfer. Based on this argument, a combination of CII- and CIII-inhibitors (malonic acid, antimycin A, myxothiazol) may yield more consistent results, although any ROS scavenged by cytochrome ''c'' may in the absence of a CIV-inhibitor result in respiratory oxygen consumption through CIV.<br />
<br />
== References ==<br />
<references/><br />
::::» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.10_TIP2k-manual&diff=149186MiPNet12.10 TIP2k-manual2018-01-11T08:46:37Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] Titration-Injection microPump. TIP2k user manual. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/f/f5/MiPNet12.10_TIP2k-Manual.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.10_TIP2k-Manual.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-03-06<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M, Gradl L, Gradl P, Gnaiger E (2014) Titration-Injection microPump. TIP2k user manual. Mitochondr Physiol Network 12.10(06):1-28.''' - [http://www.bioblast.at/index.php/File:MiPNet12.10_TIP2k-Manual.pdf Versions]<br />
<br />
The TIP2k provides unique advantages for applications which require highest accuracy of titration volumes and automatic control of complex titration-injection protocols. The TIP2k was specially developed for the O2k. The main modes of operation are automatic multi-step injections, continuous injections for steady-state respirometry, (both operated form the direct control mode), and feedback controlled operation.<br />
<br />
''Direct Control Mode:'' Operation mode for delivering a defined volume in a short pulse. A number of cycles, N, can be defined, and the duration of the interval from injection to injection determined, repeating the process N times. Several such steps can be programmed to run in sequence to give an entire TIP2k program.<br />
<br />
''Feedback Control Mode:'' Operation mode to reach and maintain a certain parameter (e.g. oxygen concentration) by automatic TIP injections.<br />
:» Product: [[TIP2k-Module]], [[Oroboros O2k-Catalogue]]<br />
|keywords=HRR, TIP2k<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=Oxygraph-2k, TIP2k, O2k-Manual, O2k-Protocol<br />
|additional=DatLab, DL6, DL7, DL6a7<br />
}}<br />
<br />
{{Technical support integrated}}<br />
<br />
<br />
__TOC__<br />
<br />
=== Cleaning TIP2k-syringes ===<br />
<br />
:::# To prevent washing out effects when the needle is inserted into the chamber the inner diameter of the TIP needle has to be very small. This means that the needle is easily blocked by small particles either precipitated from the solution or introduced with the solution. <br />
:::# While a TIP syringe has to be considered a consumable, it is possible to lengthen its lifetime by carefully following the procedures suggested in the TIP-2k Manual.<br />
:::## The main way to prevent clogging of needles is to follow rigorously the cleaning procedure after each usage. <br />
:::## Additionally, it has to be taken care that no particles are suspended in the used solutions. <br />
:::## For not very well soluble substances, filtering of the solution may be necessary. Small particles can also be introduced by laboratory activities, e.g. from paper towels or from powdered gloves. Only use underpowered gloves when working with TIP syringes!<br />
<br />
::::* Examples for proper TIP use and cleaning protocols<br />
:::# Instrumental O2 background (dithionite in phosphate buffer or MiR05)<br />
:::## Clean (fill and empty) the syringe with water.<br />
:::## Fill the syringe with dithionite solution for washing, discard immediately.<br />
:::## Fill the syringe with dithionite solution (in buffer) for use during the assay.<br />
:::## After the assay empty the syringe and immediately wash the outside of the needle with water (because there is medium sticking to the outside).<br />
:::## Wash (fill and empty cycles) the syringe three times with water.<br />
:::## Store the syringe (will be partially filled with water) or after cleaning with ethanol (then it will be partially filled with ethanol.<br />
:::# CCCP titration (CCCP dissolved in ethanol)<br />
:::## Clean (fill and empty cycle) the syringe with pure ethanol.<br />
:::## Clean with CCCP solution (in this case more important than in example 1 because you want to have a precise CCCP concentration).<br />
:::## Fill with CCCP stock solution and start the experiment.<br />
:::## After the assay empty the syringe and immediately wash the outside of the needle with water (because there is medium sticking to the outside)<br />
:::## Clean (fill and empty cycles) the syringe three times with ethanol.<br />
:::## Store the syringe (will be partially filled with ethanol) or wash it with water and then store (then it will be partially filled with water).<br />
<br />
<br />
=== TIP2k syringe blocked ===<br />
:::: The TIP syringe is harder to operate than usually.<br />
:::# Start immediately with extensive cleaning procedures. Once a needle is totally blocked it is usually impossible (with exceptions) to clean it. <br />
:::# Suitable solvents for extensive cleaning procedures depend on the substances used. <br />
:::## Chloroform was successful for some fatty acids.<br />
:::## An ultrasonic bath may be helpful in cleaning partially blocked syringes.<br />
<br />
<br />
=== Programming the TIP2k for "BG_Feedback" ===<br />
<br />
:::: In the TIP2k window the modus can be changed by clicking on either direct control or feedback control. In the feedback control several lines can be inserted, all of which take effect during the currently selected line of the main program. In the figure the implementation of program line 1 of the example set up is shown.<br />
<br />
:::: In the main window (Program line), blue highlighting shows that the first program line is selected. The first line in the feedback control window (upper right corner) sets the "start injection" value to 120 mM. The injection is only started when this condition is valid for two consecutive data points (DataN = 2). The interval is set to 200 seconds, which equals the maximum injection time when the maximum volume is set to 100 µl. These parameters are not important for the discussed purpose; however the interval has to be set to at least the (maximum) injection time calculated at the left side. Once an injection is started the program checks the following lines for "stop" instructions. If just one of these conditions is met, the injection will stop. In our example a stop condition is set at 100 µM.<br />
<br />
<br />
=== TIP2k Volume + Time ===<br />
:::: Many thanks to [[Therkelsen AIb]] from [[DK Copenhagen Quistorff B]] for pointing out this problem.<br />
<br />
::::* '''Problem:''' If the user programs an injection by setting volume and time (button "Vol+Time") the required flow is calculated by DatLab. The result is rounded to a precision that can be handled by the TIP2k. For long, slow injections this may result in significantly more or less time being necessary to inject the desired volume than the time entered by the user. If this injection is immediately or with a short delay followed by another injection this may lead to a conflict between the not finished first and the already starting second injection. Using DatLab 4 in this situation will crash the TIP program (error "TIP already running"). In DatLab 6 the program will be executed with the start of the second injection delayed. However the TIP2k status line will behave strangely in the overlapping time and events may be set at the wrong time.<br />
::::# '''Suggestions:''' Use the rounded flow as a starting point to set you own Flow + Volume or Flow + Time values that fit your experimental design.<br />
::::# '''Alternative, not tested for all DatLab versions:''' After entering the desired volume and time, press either the button "Vol+Flow" or "Flow+Time". DatLab will use the rounded flow to calculate a new time, or a new volume, respectively. <br />
<br />
<br />
== List of publications: TIP2k ==<br />
::::* [[O2k-Publications: TIP2k]]<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.11_MitoRespiration&diff=149182MiPNet12.11 MitoRespiration2018-01-11T08:43:57Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] Flux control ratios in isolated mitochondria. OXPHOS capacity and respiratory control.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/c/c9/MiPNet12.11_MitoRespiration.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.11_MitoRespiration.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-02-20<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Hand SC, Gnaiger E (2014) Flux control ratios in isolatd mitochondria. OXPHOS capacity and respiratory control in isolated mitochondria. Mitochondr Physiol Network 12.11(06):1-5.''' <br />
<br />
Methodological and conceptual features of high-resolution respirometry are illustrated in an experiment with isolated mitochondria in the Oroboros O2k. The experiment demonstrates [http://bioblast.at/images/c/c9/MiPNet12.11_MitoRespiration.pdf manual titrations] applied to study respiratory control ratios and respiratory capacity in isolated mitochondria. Application of the DatLab software is shown for real-time data analysis. The following guideline describes the experimental protocol and includes a short discussion of results. The experiments were carried out by participants of an O2k-Workshop on HRR in December 2006 (IOC36; Schröcken, Austria).<br />
<br />
The [[coupling control protocol]] (Fig. 1) was developed for respiratory studies of isolated mitochondria, and has general implications on critically assessing experiments with isolated mitochondria or in a variation of the protocol for permeabilized cells and tissues. As a substrate, succinate+rotenone or succinate without rotenone was chosen in the present example (MiPNet11.09). The experiment was aimed at assessing (1) respiratory OXPHOS and LEAK capacity, in the coupled ADP activated state and LEAK state after phosphorylation of ADP to ATP (States ''P'' and ''L''; check for possible limitations by non-saturating ADP-concentrations or loss of cytochrome c), (2) capacity of the Electron transfer-pathway (ET-pathway) in the noncoupled state (State ''E''; uncoupler titration, avoiding inhibition by high uncoupler concentrations), and (3) coupling control of OXPHOS (underestimated by the conventional RCR if the phosphorylation system is limiting).<br />
:>> Product: [[O2k-Catalogue: O2k-MultiSensor]], [[O2k-Core]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros, US LA Baton Rouge Hand SC<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Artemia, Crustaceans<br />
|preparations=Isolated mitochondria<br />
|topics=Coupling efficiency;uncoupling<br />
|couplingstates=LEAK, OXPHOS, ET<br />
|pathways=S<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-Demo, O2k-Core<br />
}}<br />
[[Image:MiPNet12.11.jpg|center|frame|Figure 1. Experiment 2006-12-14 CD-03.DLD]]<br />
<br />
<br />
== Further information ==<br />
:>> [http://www.oroboros.at/?IOC36-demo '''Excel Templates, DatLab Templates and DatLab-Demo Files'''] <br />
:>> [[Coupling control protocol]]<br />
<br />
<br />
<br />
* [[Gnaiger 2014 MitoPathways |References]]<br />
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<br />
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<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.13_Q-JunctionSCR&diff=149177MiPNet12.13 Q-JunctionSCR2018-01-11T08:41:25Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=Lemieux H, Gnaiger E (2007) MitoPathways compilation: Additive effect of succinate with substrates for Complexes I+II/Complex I. Mitochondr Physiol Network 12.13:1-5.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/c/c5/MiPNet12.13_Q-JunctionSCR_MP2.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.13_Q-JunctionSCR_MP2.pdf Versions]<br />
|year=2007<br />
|journal=Mitochondr Physiol Network<br />
|abstract=<br />
|keywords=Archive<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
|discipline=Mitochondrial Physiology<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
{{Labeling<br />
|pathways=N, S, NS<br />
|discipline=Mitochondrial Physiology<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
*[[Gnaiger 2012 MitoPathways References|References]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.15_RespiratoryStates&diff=149176MiPNet12.15 RespiratoryStates2018-01-11T08:40:56Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=Gnaiger E (2007-2011) MitoPathways: Respiratory states and flux control ratios. Mitochondr Physiol Network 12.15. - ''New edition:'' [[Gnaiger_2014_MitoPathways|Gnaiger 2014]]. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/5/5d/MiPNet12.15_RespiratoryStates.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.15_RespiratoryStates.pdf Versions]<br />
|year=2007<br />
|journal=Mitochondr Physiol Network<br />
|abstract=In [[oxidative phosphorylation]], the endergonic process of phosphorylation of ADP to ATP is coupled to the exergonic process of electron transfer to oxygen. Coupling is achieved through the proton pumps generating and utilizing the protonmotive force in a proton circuit across the inner mitochondrial membrane. This proton circuit is partially uncoupled by [[proton leak]]s. Three different meanings of uncoupling (or coupling) are distinguished by defining intrinsically [[uncoupled]], pathologically [[dyscoupled]], and experimentally [[non-coupled]] respiration. <br />
<br />
Respiratory steady states have been clearly defined by Chance and Williams (1955) according to a protocol for oxygraphic experiments with isolated mitochondria. The present state of terminology, however (e.g. [[State 2]], requires clarification, particularly for extending bioenergetics to mitochondrial respiratory physiology of the living cell.<br />
|keywords=Archive<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
|discipline=Mitochondrial Physiology<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
{{Labeling<br />
|additional=MitoPathways<br />
|discipline=Mitochondrial Physiology<br />
|articletype=Protocol; Manual, MiPNet-online Publication<br />
}}<br />
<br />
== Further information ==<br />
:>>[http://www.oroboros.at/?respiratorystates MiPNet12.15]<br />
In: Mitochondrial Pathways and Respiratory Control. Oroboros MiPNet Publ.<br />
<br />
== Abbreviations ==<br />
<br />
=== A1.1. Abbreviations for substrates ===<br />
of the [[TCA cycle]] and major entries (single capital letters for the most commonly used substrates)<br />
<br />
*P [[Pyruvate]]<br />
*G [[Glutamate]]<br />
*M [[Malate]]<br />
*S [[Succinate]]<br />
*F [[Fumarate]]<br />
*Og [[Oxoglutarate]], alpha-ketoglutarate<br />
*Ce Cellular substrates in vivo, [[endogenous]]<br />
*Cm Cellular substrates in vivo, with [[exogenous]] substrate supply from culture medium or serum<br />
<br />
=== A1.2. Other substrates and redox components of the respiratory system ===<br />
<br />
*Oca [[Octanoate]]<br />
*Paa [[Palmitic acid]]<br />
*Oct [[Octanoyl carnitine]]<br />
*Pal [[Palmitoyl carnitine]]<br />
*As [[Ascorbate]]<br />
*Tm [[TMPD]]<br />
*c [[Cytochrome c]]<br />
*Gp [[Glycerophosphate]], alpha-glycorophosphate<br />
<br />
=== A1.3. Phosphorylation system ===<br />
(adenylates, Pi, uncouplers, downstream inhibitors of ATP synthase, ANT, or phosphate) are denoted by subscripts. If Pi is always present at saturating concentration, it does not have to be indicated in the titration protocols.<br />
<br />
*Pi [[Inorganic phosphate]]<br />
*N no adenylates added (state ''L''<sub>N</sub>)<br />
*D ADP at saturating concentration (state ''P'': saturating [ADP])<br />
*D0.2 ADP at specified concentration (saturating versus non-saturating ADP is frequently not specified in [[State 3]])<br />
*T ATP (state ''L''<sub>T</sub>)<br />
*TD ATP+ADP (state ''P'', in the presence of physiological high (mM) ATP concentrations)<br />
*T[ADP] High ATP and varying ADP concentrations, in the range between states T and TD.<br />
*0my [[Oligomycin]] (state ''L''<sub>Omy</sub>)<br />
*Atr Atractyloside (state ''L''<sub>Atr</sub>)<br />
*u [[Uncoupler]] at optimum concentration for maximum non-coupled flux (state ''E'').<br />
<br />
=== A1.4. Inhibitors ===<br />
of respiratory complexes, dehydrogenases or transorters:<br />
<br />
*Ama [[Antimycin A]]<br />
*Azd Sodium [[azide]]<br />
*Hci [[Hydroxycinnamate]]<br />
*Kcn [[Cyanide]]<br />
*Mna [[Malonic acid]]<br />
*Myx [[Myxothiazol]]<br />
*Rot [[Rotenone]]<br />
<br />
=== A1.5. Respiratory states and flux control ratios ===<br />
<br />
Coupling control states<br />
<br />
*''E'', [[Electron transfer-pathway]] capacity state<br />
*''L'', [[LEAK state]]<br />
*''P'', [[OXPHOS capacity]] state<br />
*''R'', [[ROUTINE]] state of cell respiration<br />
<br />
Coupling control ratios (''CCR'')<br />
<br />
*''L/E'', [[LEAK]] ''CCR''<br />
*''P/E'', [[Phosphorylation system]] capacity ''CCR''<br />
*''R/E'', [[ROUTINE]] ''CCR''<br />
*(''R-L'')/''E'', [[netROUTINE]] ''CCR''<br />
<br />
== References ==<br />
<br />
*[[Gnaiger_2009_Int J Biochem Cell Biol|Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837–1845.]]<br />
*[[Pesta 2012 Methods Mol Biol|Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.]]<br />
*[[Gnaiger 2012 MitoPathways References|References]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.17_BovineHeartMito&diff=149172MiPNet12.17 BovineHeartMito2018-01-11T08:38:43Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]Mitochondrial respiration with frozen bovine heart mitochondria: diagnostic tests.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/7/74/MiPNet12.17_FrozenBovineHeartMito.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.17_FrozenBovineHeartMito.pdf Versions]<br />
|authors=Oroboros<br />
|year=2011-12-11<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Petrosyan S, Naviaux R, Haas RH, Gnaiger E (2011) Mitochondrial respiration with frozen bovine heart mitochondria: diagnostic tests. Mitochondr Physiol Network 12.17(04).''' <br />
<br />
The following demo experiment was performed in preparation for the lecture hall experiment, presented at the Mitochondrial Medicine conference (Diagnosis of Disease: Workshop on HRR at Mitochondrial Medicine 2007, UMDF, San Diego, USA, June 2007 (IOC40). For explanation of symbols, see [[Gnaiger 2012 MitoPathways]].<br />
<br />
[[Image:MiPNet12.17.jpg|center|100px|frame|Figure 1. Simultaneous display of oxygen concentration (blue lines) and oxygen flux (respiratory rate, red lines; negative time derivative of oxygen concentration) in chamber A and B.]]<br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros <br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Bovines<br />
|tissues=Heart<br />
|preparations=SMP<br />
|topics=Coupling efficiency;uncoupling, Cyt c<br />
|couplingstates=OXPHOS<br />
|pathways=S<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-Demo, O2k-Core<br />
}}<br />
== Further information ==<br />
:'''>> [[MiPNet12.17|Excel Templates and DatLab-Demo Files]]'''<br />
<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.20_O2k-calibration_tutorial&diff=149169MiPNet12.20 O2k-calibration tutorial2018-01-11T08:37:03Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k: Oxygen calibration and O2k-background tutorial. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/b/b5/MiPNet12.20_O2k-CalibrationTutorial.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.20_O2k-CalibrationTutorial.pdf Versions] |authors=Oroboros<br />
|year=2010-10-19<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Gnaiger E (2006) O2k: Oxygen calibration and O2k-background tutorial. Mitochondr Physiol Network 12.20.''' » <br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-SOP<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet12.23_FiberRespiration&diff=149167MiPNet12.23 FiberRespiration2018-01-11T08:36:22Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/49/MiPNet12.23_FibreRespiration.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-07-19<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Doerrier C, Lemieux H, Votion DM, Gnaiger E (2007-16) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(08 in prep):1-8.''' <br />
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the Oroboros O2k. The experiment demonstrates [http://bioblast.at/images/e/ef/MiPNet09.12_O2k-Titrations.pdf manual titrations] applied to study respiratory capacity in permeabilized fibres. Application of the [[DatLab]] software is shown for real-time data analysis (compare [[MiPNet12.09]]). The following guideline describes the [[1GM;2D;3S;4U;5Rot-]] SUIT protocol and includes a short discussion of results (see [[Votion 2012 PLoS One]]). The experiment was carried out by participants of an O2k-Workshop on HRR in December 2007 ([[MiPNet12.24 IOC44 |IOC44]]; Schroecken, Austria).<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros, BE_Liege_Votion DM<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Horse<br />
|tissues=Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
|topics=Coupling efficiency;uncoupling, Cyt c<br />
|couplingstates=LEAK, OXPHOS, ET<br />
|pathways=N, S, NS<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-Demo, O2k-Core, 1GM;2D;3S;4U;5Rot-<br />
}}<br />
== SUIT protocol ==<br />
[[File:1GM;2D;2c;3S;4U;5Rot;6Ama.jpg |right|300px|link=1GM;2D;3S;4U;5Rot-]]<br />
****: SUIT protocol: [[1GM;2D;3S;4U;5Rot-]]<br />
<br />
'''SUIT states:''' 1-2[[GM]] 3-4[[GMS]] 5[[S]] 6[[ROX]]<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
! Step<br />
! Respiratory state<br />
! Pathway control<br />
! Entry through electron transfer Complexx<br />
<br />
|-<br />
| 1GM<br />
| [[GM]](L)<br />
| N<br />
| CI<br />
<br />
|-<br />
| 2D<br />
| [[GM]](P)<br />
| N<br />
| CI<br />
<br />
|-<br />
| 2c<br />
| [[GM]](P)<br />
| N<br />
| CI<br />
<br />
|-<br />
| 3S<br />
| [[GMS]](P)<br />
| NS<br />
| CI<small>&</small>II<br />
<br />
|-<br />
| 4U<br />
| [[GMS]](E)<br />
| NS<br />
| CI<small>&</small>II<br />
<br />
|-<br />
| 5Rot<br />
| [[S]](E)<br />
| S<br />
| CII<br />
<br />
|-<br />
| 6Ama<br />
| ROX<br />
<br />
|}<br />
<br />
[[Image:MiPNet12.23.jpg|center|800px]]<br />
Figure 1. Oxygen concentration ([μM] blue line) and oxygen flux per mg wet weight of muscle ([pmol∙s<sup>-1</sup>∙mg<sup>-1</sup>] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol.<br />
<br />
A multiple substrate-uncoupler-inhibitor titration protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The experiment was aimed at an assessment of a sequence of titrations, inducing stepwise defined respiratory states.<br />
<br />
<br />
<br />
:::#<span style="color:red"> '''LEAK state with type N substrates, N''<sub>L</sub>'':'''</span> Non-phosphorylating resting state with NADH-linked (type N) substrates glutamate&malate (GM; without adenalytes; CI-linked pathway to Q).<br />
:::#<span style="color:green">'''OXPHOS capacity with type N substrates, N''<sub>P</sub>'':'''</span> Respiratory capacity in the active coupled state (GM with ADP).<br />
:::#<span style="color:black">'''Cytochrome ''c'' test for quality control:'''</span> Further addition of cytochrome ''c'' yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome ''c'' would be indicated by a stimulation of respiration).<br />
:::#<span style="color:green">'''OXPHOS capacity with type NS substrates (CI<small>&</small>II-linked pathway to Q), NS''<sub>P</sub>'':'''</span> Respiratory stimulation by convergent electron flow through Complexes I<small>&</small>II at the Q-junction, in the coupled state after further addition of succinate (S), as an estimate of OXPHOS capacity with reconstitution of the TCA cycle ([[Gnaiger 2009 Int J Biochem Cell Biol]]).<br />
:::#<span style="color:blue">'''Electron transfer-pathway (ET-pathway) capacity with type NS substrates, NS''<sub>E</sub>'':'''</span> Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ET capacity by the phosphorylation system.<br />
:::#<span style="color:blue">'''ET capacity with type S (CII) substrate, S''<sub>E</sub>'':'''</span> ET capacity with succinate, after blocking Complex I with rotenone.<br />
:::#'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (inhibitor of Complex III).<br />
<br />
<br />
== Reference ==<br />
<br />
::::* Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7: e34890. - [[Votion 2012 PLoS One |Open Access]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet14.01_ESD-damage&diff=149166MiPNet14.01 ESD-damage2018-01-11T08:34:55Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=http://wiki.oroboros.at/index.php/O2k-Manual|O2k-Manual]] Electrostatic discharge (ESD): damage and protection. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/5/50/MiPNet14.01_ESD_Damage.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet14.01_ESD_Damage.pdf Versions]<br />
|authors=<br />
|year=2011<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M (2011) Electrostatic discharge (ESD): damage and protection. Mitochondr Physiol Network 14.01(07).''' - <br />
<br />
''This is an old version; updated in:'' '''[[MiPNet07.08 User information]]'''<br />
|keywords=<br />
|mipnetlab=<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet14.05_TPP-mtMembranePotential&diff=149163MiPNet14.05 TPP-mtMembranePotential2018-01-11T08:33:56Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] TPP+ and mt-membrane potential.<br />
|info=[[File:PDF.jpg|100px|link=http://www.bioblast.at/images/7/78/MiPNet14.05_TPP-MitoMembranePotential.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet14.05_TPP-MitoMembranePotential.pdf Versions]<br />
|authors=Oroboros<br />
|year=2009-01<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Sumbalova Z, Fasching M, Gnaiger E (2009) TPP+ and mt-membrane potential. Mitochondr Physiol Network 14.05.'''<br />
<br />
[[Image:MiPNet14.05_TPP-MitoMembranePotential.png|center|1200px|Sumbalova Z, Fasching M, Gnaiger E (2011) Substrate control in mitochondrial respiration and regulation of mitochondrial membrane potential. Abstract Mitochondrial Medicine Chicago.]]<br />
<br />
[[Sumbalova_2011Abstract_Mitochondrial_Medicine |Abstract: Sumbalova Z, Fasching M, Gnaiger E (2011) Substrate control in mitochondrial respiration and regulation of mitochondrial membrane potential. Abstract Mitochondrial Medicine Chicago.]]<br />
<br />
[[Media:MiPNet14.05_TPP-MitoMembranePotential.pdf|'''previous version''']]; <br />
MiPNet14.05 Appendix: Mathematics for mitochondrial membrane potential: [[File:MiPNet14.05 App TPP-Mathematics-MembranePotential.pdf]]<br />
<br />
<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|organism=Mouse<br />
|tissues=Nervous system<br />
|preparations=Homogenate, Isolated mitochondria<br />
|instruments=Oxygraph-2k, TPP, O2k-Protocol<br />
|additional=O2k-Demo, O2k-MultiSensor<br />
}}<br />
== Further information ==<br />
:>> [[O2k-Protocols| '''Excel Templates, DatLab Templates and DatLab-Demo Files''']]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet14.06_Instrumental_O2_background&diff=149162MiPNet14.06 Instrumental O2 background2018-01-11T08:33:22Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf |Bioblast pdf]] » [http://www.bioblast.at/index.php/File:MiPNet14.06_InstrumentalO2Background.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-02-09<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M, Gnaiger E (2016) O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. Mitochondr Physiol Network 14.6(05):1-8.''' <br />
<br />
'''O2k-Protocols SOP''': Correction for instrumental background oxygen flux is a standard in high-resolution respirometry, automatically performed by DatLab. Background measurements provide a test of instrument function. In the Oroboros O2k, background corrections are usually within a few % of experimental flux over the entire experimental oxygen range. At minimum activities, however, even the small background effects become highly significant and require compliance to standard operating procedures (O2k-SOP) described in this chapter as part of the [[MitoFit Quality Control System]]. This is part 2 of O2k Quality Control.<br />
<br />
:» O2k-Protocols SOP: O2k Quality Control 1 »[[MiPNet06.03 POS-calibration-SOP]]«<br />
:» O2k-Manual »[[MiPNet19.18E O2 flux analysis]]«<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]<br />
|keywords=Instrumental background<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
__TOC__<br />
<br />
[[File:Expand.png|right|45px |Click to expand or collaps]]<br />
<div class="toccolours mw-collapsible mw-collapsed"><br />
:::[[File:Questions.jpg|left|24px |Explanation of terms]] '''» Keywords'''<br />
<div class="mw-collapsible-content"><br />
::: '''Instrument'''<br />
::::» [[O2k-FluoRespirometer]]<br />
::::» [[DatLab]]<br />
::::» [[O2k-Chamber]]<br />
::::» [[MiPNet19.18E O2 flux analysis]] |O2k-Manual: O2 flux analysis]]<br />
::::» [[MiPNet19.03 O2k-cleaning and ISS |O2k-Protocols SOP: O2k-cleaning]]<br />
<br />
::: '''Concept'''<br />
::::» [[Concentration]]<br />
::::» [[High-resolution respirometry]]<br />
::::» [[Instrumental background oxygen flux]]<br />
::::» [[MiPNet14.06 Instrumental O2 background |O2k-Protocols SOP: Instrumental O2 background]]<br />
::::» [[MitoFit Quality Control System]]<br />
::::» [[O2k-SOP |O2k-Protocols SOP]]<br />
::::» [[Oxygen |Oxygen, dioxygen, O<sub>2</sub>]]<br />
</div><br />
</div><br />
<br />
{{Technical support integrated}}<br />
<br />
::::* First step - O2k Quality Control 1: »[[MiPNet06.03 POS-calibration-SOP]]«<br />
[[File:O2k-QCS.jpg|350px|O2k-QCS]] <br />
<br />
::::* Second step - O2k Quality Control 2<br />
<br />
[[Image:MiPNet14.06.jpg|frame|Instrumental background experiment, measuring oxygen flux without biological sample at four oxygen levels (left), and linear relation between instrumental background oxygen flux and oxygen concentration (right). Modified after: Gnaiger E (2001).]]<br />
== Standard operating procedure ==<br />
:::: If all quality control criteria of the O2 sensor test are met, the operator can be assured that the quality of the sensor signal is acceptable. Next, the quality of the O2k-Chamber assembly has to be tested, described in detail as an O2k-SOP:<br />
:::: The '''O2k-chamber test''' provides quality control at an instrumental level beyond the O2 sensor test:<br />
::::# [[O2k-Chamber]] not properly assembled or broken.<br />
::::## [[OroboPOS-Holder]] not properly positioned.<br />
::::## [[O2k-Chamber Holder]] not properly positioned; V- and O-rings not properly mounted ([[V-ring\30-35-4.5 mm]], [[O-ring\Viton\18x2 mm]]).<br />
::::# [[Volume-Calibration Ring]] not properly positioned by chamber volume calibration.<br />
::::# [[O-ring\Viton\12x1 mm]] injured and must be replaced on the stopper.<br />
::::# [[Stopper\black PEEK\conical Shaft\central Port]] broken conical edge or O-ring not properly applied. <br />
::::# [[OroboPOS-Seal Tip]] leaky.<br />
::::# Experimental medium consumes oxygen due to microbial contamination.<br />
<br />
::::* Next step - when measuring cytochrome ''c'' oxidase activity: Autoxidation of ascorbate and TMPD causes a [[MiPNet06.06_ChemicalBackground |chemical background oxygen flux]]. DatLab provides real-time correction for instrumental and chemical background.<br />
<br />
<br />
== Trouble shooting ==<br />
:::'''If specifications given in the [[MiPNet14.06 Instrumental O2 background |Instrumental O<sub>2</sub> background]] are not obtained''': check components for locating the problem.<br />
::::# Check the stirring bars for any contamination.<br />
::::# Check the stoppers for the quality of the O-rings and the conical edges.<br />
::::# If no indications of a defect are observed, disassemble the O2k-chamber.<br />
::::# Check the [[glass chamber]] for contamination or for broken edges.<br />
::::# Clean the copper block of the O2k and reassemble the O2k-chamber.<br />
::::# Reassemble and clean the chambers: [[MiPNet19.03 O2k-cleaning and ISS]].<br />
::::# Perform an O2 sensor test and - if successful - an O2k-chamber test, using fresh incubation medium.<br />
::::# If the problem with the instrumental O<sub>2</sub> background remains in one chamber, switch stoppers between chambers A and B.<br />
::::# Perform an O2k-chamber test (the sensor test is not necessary).<br />
::::# If the problem with the instrumental O<sub>2</sub> background remains in the same chamber, switch glass chambers between the left and right side of the O2k.<br />
::::# Perform an O2 sensor test and - if successful - proceed with the O2k-chamber test.<br />
::::# If the problem with the instrumental O<sub>2</sub> background remains in the same chamber, switch sensors between the left and right chamber.<br />
<br />
<br />
=== Instrumental oxygen background test for permeabilized muscle fibres ===<br />
<br />
:::# While biopsy sampling and fibre preparation proceed: Perform air calibration in [[MiR06Cr]], then close the chamber to evaluate instrumental background at air saturation (c. 10 min): This is a quality control of the medium, important under field conditions, where medium preservation (sterility) may be less controlled than in the lab. <br />
:::# Elevate oxygen concentration to 450-500 µM with oxygen gas ([[Syringe\60 ml\Gas-Injection]]), close and after two to three min perform a [[stirrer test]] (using the automatic stirrer test function of DatLab). This is important, since the [[OroboPOS]] may have a different response time at elevated oxygen concentration. If the response time increases dramatically, then the sensor may even show a non-linear response to oxygen concentration at high oxygen levels. <br />
:::# Instrumental background: After 20 min, open the chamber and allow O<sub>2</sub> to drop to c. 350 µM, close for 20 min, open and drop O<sub>2</sub> to c. 250 µM (this should be the lowest experimental O<sub>2</sub> concentration). <br />
:::# Increase O<sub>2</sub> with H<sub>2</sub>O<sub>2</sub> injection (c. 2 µl) to 400 µM, measure for 15-20 min instrumental background, simulating a re-oxygenation during the experiment.<br />
:::# Increase O<sub>2</sub> with H<sub>2</sub>O<sub>2</sub> injection (c. 1 µl) to 450-500 µM, until the fibres are added, for equilibrating the instrument at high O<sub>2</sub>.<br />
:::# Addition of permeabilized fibres into the O2k-Chamber: » [[Permeabilized muscle fibres]].<br />
<br />
<div id="Instrumental O2 DLP anchor"></div><br />
=== Apparent oxygen flux in closed chamber near air saturation with pure medium ===<br />
<br />
:::: The uncorrected slope of the oxygen signal over time in a [[Talk:Closed_chamber|closed chamber]] at air-calibrated oxygen concentration is an important control parameter. It reflects the consumption of oxygen by the [[polarographic oxygen sensor]] ([[POS]]). The theoretical value is calculated by DatLab in the O2 Calibration window (Supplement C in [[MiPNet06.03 POS-calibration-SOP]]). The theoretical value at 37 °C (O2 slope uncorrected) is usually between 2 and 3 pmol·s<sup>-1</sup>·ml<sup>-1</sup>. The actual values should correspond very closely to the expected slope, i.e. within ± 1 pmol·s<sup>-1</sup>·ml<sup>-1</sup>.<br />
::::* Values higher than 4-5 pmol·s<sup>-1</sup>·ml<sup>-1</sup> at 37 °C may therefore indicate a [[Talk:Biological_contamination|biological contamination]] in the chamber or in the medium.<br />
::::* Lower values may indicate: <br />
::::# Air bubbles in the closed chamber: switch on the internal illumination of the O2k and inspect the chamber through the front window. Remove any air bubbles.<br />
::::# A large volume of medium collected in the receptacle of the stopper: siphon off any medium.<br />
::::# A larger chamber volume: check [[O2k-Chamber volume calibration]].<br />
::::# If an O2k-MultiSensor stopper with multiple ports is used, it is particularly important to siphon off excess liquid from top of stopper. Any convection of liquid must be avoided, which otherwise results in an apparently leaky chamber. <br />
<br />
<br />
<br />
== Instrumental O2 DLP ==<br />
<br />
Instrumental O2 [[DL-Protocols]] (DLP) are used for calibrations and instrumental quality control, typically without experimental sample in the incubation medium.<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
! DL-Protocol<br />
! Description<br />
! DLP File<br />
<br />
|-<br />
| Instrumental O2 background TIP2k <br />
| Instrumental O2 background with TIP2k including OroboPos test and two-point calibration <br />
| [[Media:Instrumental O2 background_TIP2k.DLP|Instrumental O2 background TIP2k.DLP]] <br />
|-<br />
| Instrumental O2 background manual injections<br />
| Instrumental O2 background with manual injections including OroboPos test and two-point calibration<br />
| [[Media:Instrumental_O2_background_manual_injections.DLP|Instrumental O2 background manual injections.DLP]]<br />
|-<br />
| Instrumental high O2 background TIP2k <br />
| Instrumental high O2 background with TIP2k including OroboPos test and two-point calibration<br />
| [[Media:Instrumental_high_O2_background_TIP2k.DLP|Instrumental high O2 background TIP2k.DLP]]<br />
|-<br />
| Instrumental high O2 background manual injections<br />
| Instrumental high O2 background with manual injections including OroboPos test and two-point calibration<br />
| [[Media:Instrumental_high_O2_background_manual_injections.DLP|Instrumental high O2 background manual injections.DLP]]<br />
|} <br />
<br />
A full list of DL-Protocols (Instrumental DL-Protocols and SUIT DL-Protocols) is displayed in [[DL-Protocols]].<br />
<br />
<br />
== DatLab-Analysis templates and DatLab Demo Files ==<br />
<br />
[[File:2017-05-17 P2-01 Instrumental O2 background TIP2k.jpg|400px|left]]<br />
<br />
<br />
:: '''DatLab 7:''' [[Media:O2 background.xlsx|Instrumental O2 background.xlsx]]<br />
<br />
:: '''DatLab 6:''' [[Media:O2k-Background DL6.xlsx|O2k-Background.xlsx]]<br />
<br />
:: '''DatLab 5:''' [[Media:O2k-Background DL5.xlsx|O2k-Background.xlsx]]<br />
<br />
<br />
:: '''Demo file:''' <br />
::: [[Media:MiPNet14.06 2014-07-24 P4-02 Instr-background.DLD|MiPNet14.06_2014-07-24_P4-02_Instr-background.DLD]] (norm. oxygen)<br />
::: [[Media:MiPNet10.04 2014-02-20 P4-02 O2-calib high-O2.DLD|MiPNet10.04_2014-02-20_P4-02_ O2-calib high-O2.DLD]] (high oxygen)<br />
<br />
== References ==<br />
<br />
:::* Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96. - [[Gnaiger_1995_J_Bioenerg_Biomembr |»Bioblast link«]]<br />
:::* Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. - [[Gnaiger_2001_Respir_Physiol |»Bioblast link«]]<br />
:::* Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - [[Gnaiger_2008_POS |»Bioblast link«]]<br />
<br />
<br />
<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-SOP, DatLab<br />
}}<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet14.10_O2k-Top_10&diff=149160MiPNet14.10 O2k-Top 102018-01-11T08:32:34Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:Logo OROBOROS INSTRUMENTS.jpg|right|60px|link=OROBOROS INSTRUMENTS|OROBOROS]] Oroboros (2017) Top 10 reasons for Oroboros Instruments. Mitochondr Physiol Network 14.10(09):1-4. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/44/MiPNet14.10_O2k-Top10.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:MiPNet14.10_O2k-Top10.pdf Versions]<br />
|authors=Oroboros<br />
|year=2017-01-03<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''We summarize 10 compelling reasons for chosing the Oroboros O2k, for collaborating with Oroboros Instruments, and for spreading the good news of the Oroboros MiPNet. ‘Top 10’ reflects our corporate goals as a network rather than a linear sequence. The numbering, therefore, is largely arbitrary and does not express priorities.''' <br />
|keywords=O2k, HRR, Gentle Science, Cooperation and Feedback in Science, CSR<br />
|mipnetlab=AT Innsbruck Oroboros<br />
|articletype=MiPNet-online Publication<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods, mt-Awareness<br />
}}<br />
<br />
<br />
[[File:O2k-Fluorometer Rollup.jpg|130px|O2k-Fluorometer]] [[File:O2k-Network.png|140px]] [[File:O2k-Workshops.png|170px]] [[File:O2k-Fluorometer.JPG|140px| O2k]] [[File:O2k-Countries.jpg|160px]] [[Image:OROBOROS-solo.png|140px]] [[File:Odra Noel Powerplant.jpg|130px]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet14.12_QA-Workshop&diff=149158MiPNet14.12 QA-Workshop2018-01-11T08:32:03Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-WorldWide.jpg|right|60px|link=http://www.bioblast.at/index.php/MiPNet_Reference_Laboratories|MiPNet Reference Laboratories]] Quality assurance in respirometric diagnosis of mitochondrial function. MiPNet Workshop 9-10 Dec 2009. Mitochondr Physiol Network 14.12: 1-13.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/97/MiPNet14.12_QA-Workshop_2009.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet14.12_QA-Workshop_2009.pdf Versions]<br />
|authors=OROBOROS<br />
|year=2009<br />
|journal=Mitochondr Physiol Network<br />
|abstract=<br />
|keywords=MitoPathways<br />
|mipnetlab=AT Innsbruck OROBOROS <br />
|articletype=Workshop, MiPNet-online Publication<br />
|discipline=Mitochondrial Physiology, Biomedicine<br />
}}<br />
{{Labeling|articletype=Workshop, MiPNet-online Publication<br />
|discipline=Mitochondrial Physiology, Biomedicine<br />
}}<br />
<br />
__TOC__<br />
<br />
==Towards Quality Assurance==<br />
<br />
Quality Assurance (QA) is too big a term for a small workshop on diagnosis of mitochondrial (mt) respiratory function, even when the focus is restricted to the model of permeabilized muscle fibres. There are several milestones in the quest '''towards QA''', including continuous quality control and incorporating ‘Best Practice’ in the laboratory. We are well aware of the fact that '''in practice''' we may aim at '''better''' standards, but will hardly ever establish any final '''best'''. QA in general may set impractically high demands on a laboratory, hence the '''required level of QA depends on the specific application'''. QA will be different in (i) an experimental study on mt-respiratory function of mouse skeletal muscle, (ii) a comparison of aged humans versus young controls using biopsies, or (iii) the diagnosis of a mt-myopathy in a single patient when an individual is evaluated (she/he really cares) versus a (matched – science has to care) control group. <br />
<br />
<br />
Against this background, we can reach several unambiguous conclusions:<br />
<br />
::1. '''Some level of QA is required''' in all studies on mt-respiratory function – from basic science to clinical applications.<br />
<br />
::2. '''Sharing the practical expertise''' of different research groups in this rapidly expanding field provides an effective and economical approach towards higher standards of science by implementing QA.<br />
<br />
::3. '''Multiple benefits''' will result from the development and application of appropriate standards of QA - for the individual patient (very few laboratories are involved), the individual scientist (all of us, all of our collaborators), the laboratory, mt-respiratory physiology in terms of scientific reputation (including some companies with positive or negative impacts), the particular segment of our health care system.<br />
<br />
The benefits and implications are potentially enormous, considering the '''impact of mitochondrial medicine on the quality of life''': life style related to exercise and nutrition, obesity, degenerative diseases, metabolic syndrome, muscular and cognitive dysfunction, rare mt-diseases, healthy aging, cardiovascular diseases, reducing the risks for a range of cancers, immunological fitness, competitive and noncompetitive sports, hypoxia, ischemia-reperfusion, ...<br />
<br />
<br />
It is far more exciting to discuss these challenging topics than to talk about quality control of the chemicals used as inhibitors or in mitochondrial respiration media, tissue wet weight or dry weight measurements, or about valid estimates of the proton leak in a respirometric protocol. A focus on QA, however, helps to eliminate unreliable data which are a source of confusion, lead to unnecessary controversies, or even overshadow the potential of accurate diagnosis of a disease. Explicit implementation of QA will provide a basis of increased '''recognition and reputation of our field of research''', and may be considered as an expression of '''corporate social responsibility within the mitochondrial physiology network'''. This MiPNet QA-Workshop includes but is not restricted to instrumental performance, and presentations are invited to cover not only applications of the OROBOROS Oxygraph-2k but also extend the discussion to other instruments<br />
<br />
==Aims==<br />
<br />
<br />
The topics and aims of the proposed workshop on ‘Respirometry of permeabilized muscle fibres: towards quality assurance in the diagnosis of mitochondrial function’ are:<br />
<br />
::1. '''Presentation of presently available components of QA''': methodological details, exchange of laboratory protocols with updates.<br />
<br />
::2. '''Science sessions''': protocols, traces, results, concepts, perspectives.<br />
<br />
::3. '''Summary of practically important segments and perspectives of QA'''.<br />
<br />
<br />
Is it worthwhile considering a joint project application? Can you come up with specific suggestions? Some of the workshop participants will attend the subsequent ''Course on High-Resolution Respirometry'' (IOC54; Schröcken, Austria), during which we might complete and extend the summary from the workshop in a small group.<br />
<br />
<br />
<br />
<br />
== Participants ==<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
!<br />
! Participant<br />
! Institution<br />
|-<br />
|<br />
|[[Bilet L|Bilet Lena]]<br />
|'''[[NL Maastricht Schrauwen P]]''': Maastricht University (NL)<br />
|-<br />
|[[File:Boushel.jpg|80px|Boushel Robert C]]<br />
|[[Boushel RC|Boushel Robert C]]<br />
|'''[[DK Copenhagen Dela F]]''': University of Copenhagen (DK)<br />
|-<br />
|[[File:Martin.jpg|80px|Burtscher Martin]]<br />
|[[Burtscher M|Burtscher Martin]]<br />
|'''[[AT Innsbruck Burtscher M]]''': Univ. Innsbruck (AT)<br />
|-<br />
|<br />
|'''Dela Flemming'''<br />
|'''Faculty of Health Sciences''' (DK)<br />
|-<br />
|[[File:Mario.jpg|80px|Mario Fasching]]<br />
|[[Fasching Mario]]<br />
|'''[[AT Innsbruck OROBOROS]]'''<br />
|-<br />
|[[File:Garcia-RovesP.jpg|80px|Garcia-Roves Pablo Miguel]]<br />
|[[Garcia-Roves PM|Garcia-Roves Pablo Miguel]]<br />
|'''[[SE Stockholm Morein T]]''': Karolinska Institute (SE)<br />
|-<br />
|[[File:Gnaiger Erich.jpg|80px|Erich Gnaiger]]<br />
|[[Gnaiger Erich]]<br />
|'''[[AT Innsbruck OROBOROS]]'''<br />
|-<br />
|<br />
|[[Haberberger Birgit]]<br />
|'''Institut für Humangenetik''' (DE)<br />
|-<br />
|<br />
|[[Hansson Magnus J]]<br />
|'''[[SE Lund Elmer E]]''': Lund University (SE)<br />
|-<br />
|[[File:David.jpg|80px|Harrison David K]]<br />
|[[Harrison DK| Harrison David K]]<br />
|'''[[AT Innsbruck OROBOROS]]'''<br />
|-<br />
|<br />
|[[Hoeks J|Hoeks Joris]]<br />
|'''[[NL Maastricht Schrauwen P]]'''<br />
|-<br />
|<br />
|[[Huettenhofer A|Huettenhofer Alexander]]<br />
|'''Innsbruck Medical University''' (AT)<br />
|-<br />
|[[File:Iyer.jpg|80px|Iyer Shilpa]]<br />
|[[Iyer S|Iyer Shilpa]]<br />
|'''[[US AR Fayetteville Iyer S]]''': Fulbright College of Arts and Sciences (US)<br />
|-<br />
|<br />
|[[Janke L|Janke Linda]]<br />
|'''[[DE Duesseldorf Roden M]]''': Heinrich Heine University Düsseldorf (DE)<br />
|-<br />
|[[File:Kane DA.jpg|80px]]<br />
|[[Kane DA|Kane Daniel A]]<br />
|'''[[CA Antigonish Kane DA]]''': St. Francis Xavier University (CA)<br />
|-<br />
|<br />
|[[Kraunsoee R|Kraunsoee Regitze]]<br />
|'''[[DK Copenhagen Dela F]]''': University of Copenhagen (DK)<br />
|-<br />
|<br />
|[[Kruse S| Kruse Shane]]<br />
|'''[[US WA Seattle Marcinek DJ]]''': University Washington Med Center (US)<br />
|-<br />
|}<br />
<br />
<br />
== Summary ==<br />
<br />
:*The workshop group wants to continue this discussion and move towards a more formal QA-approach, preferentially forming a working group within the [[MiPsociety]], and linking to other societies. A circular should be sent to all MiPmembers, with the summary of the QA workshop, future perspectives, and questions on feedback. A special evening session might be devoted to QA at [[MiP2010]] (but will there be sufficient time?).<br />
<br />
:*'''QA working group:''' The QA workshop and related correspondence showed that there is a critical mass for a working group; avoid forming a closed group of ‘insiders’, remain open but focused. The special topic of respirometry of permeabilized muscle fibres appears to be adequate and manageable as a first step, whereas the general correspondence suggest a broader perspective (including isolated mitochondria, intact and permeabilized cultured and primary cells) for future QA workshops.<br />
<br />
:*'''Publication:''' Methods paper on the basis of a series of QA workshops; possible key words are:<br />
:::* Methods of taking biopsies<br />
:::* Sample handling and sample preservation<br />
:::* Respirometric instruments<br />
:::* Experimental conditions (temperature; media; controls incl. cyt c test; oxygen levels)<br />
:::* Wet weight/dry weight; normalization of respirometric flux<br />
:::* Mechanical separation of fibres<br />
:::* Permeabilization of fibres (concentration of sponin, time, temperature, washes)<br />
:::* Substrate concentrations (how to evaluate correct or optimal concentration; stock solution preparation)<br />
:::* Miminal protocol (evaluation; minimal protocol for comparison and quality control)<br />
:::* Pitfalls<br />
:::* Exclusion criteria<br />
:::* The methods paper should include a meta-analysis of published data<br />
<br />
:*'''QA website:''' Place for [[O2k-Protocols|protocols]], study design, information for researchers on groups who are doing similar work.<br />
<br />
:*'''Towards QA''' is a theme of [[Gentle Science]].<br />
<br />
== Impressions ==<br />
<br />
<br />
<gallery mode=default perrow=3 widths="300px" heights="300px"><br />
File:QA-Workshop 01.JPG<br />
File:QA-Workshop 02.JPG<br />
File:QA-Workshop 03.JPG<br />
</gallery><br />
<br />
<br />
<br />
== Programme==<br />
'''» [[File:MiPNet14.12 QA-Workshop-2009 Program.pdf]]'''</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet14.13_Medium-MiR06&diff=149155MiPNet14.13 Medium-MiR062018-01-11T08:30:36Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Mitochondrial respiration medium - MiR06. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/d/d9/MiPNet14.13_Medium-MiR06.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet14.13_Medium-MiR06.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-08-30<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M, Fontana-Ayoub M, Gnaiger E (2016) Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13(06):1-4.''' <br />
<br />
Mitochondrial respiration medium MiR06 was developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06+creatine.<br />
:» Product: [[MiR05-Kit]]<br />
|keywords=MiR06<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=O2k-Protocol<br />
|additional=O2k-chemicals and media<br />
}}<br />
== Application of MiR06 in [[HRFR]] ==<br />
<br />
'''MiR06: Mitochondrial Respiration Medium''' ([[#Preparation of MiR05 (MiR06) stock solution|MiR06]] = [[MiR05]] + [[Catalase]]).<br />
<br />
::Oxygen solubility factor in MiR05 or MiR06 at 30 °C and 37 °C = 0.92<br />
<br />
::pH of MiR05/06: 7.2 (20 °C), 7.2 (25 °C), 7.1 (30 °C), 7.1 (35 °C), 7.0 (37 °C)<br />
<br />
<br />
== Re-oxygenation with H<sub>2</sub>O<sub>2</sub> titrations ==<br />
<br />
An experiment needs not necessarily be terminated , because of running out of oxygen. There are different possibilities to re-oxygenate.<br />
* '''To increase oxygen levels''' small volumes (µl) of [[Reoxygenation#Preparation_of_200_mM_H2O2_stock_solution|200 mM H<sub>2</sub>O<sub>2</sub> stock solution]] are injected into the O2k-chamber filled with 2 ml MiR06.<br />
<br />
With MiR06 (or [[MiR06Cr]]), the medium in the O2k-chamber can be re-oxygenated very conveniently with H<sub>2</sub>O<sub>2</sub> titrations. The initial increase in oxygen, however, is preferentially made with oxygen gas, since there is the risk of bubble formation if the oxygen concentration is increased in a single large step. If oxygen gas is not available for the initial oxygenation, a very small bubble may be left in the chamber while slowly rising the oxygen level to 500 µM with additions of H<sub>2</sub>O<sub>2</sub>, such that gas can escape into the small bubble and then be extruded by fully closing the chamber. During the experiment, re-oxygenations are sufficiently small such that H<sub>2</sub>O<sub>2</sub> titrations into the closed chamber do not lead to gas bubble formation.<br />
<br />
*'''Re-oxygenation by adding H<sub>2</sub>O<sub>2</sub> to a catalase containing medium (Medium-MiR06)'''<br />
<br />
:Add catalase at a final concentration of 280 IU/ml to the medium at the beginning of an experiment. When oxygen starts to become limited, inject 1-3 µl of an approximately 200 mM H2O2 stock solution. The H<sub>2</sub>O<sub>2</sub> will immediately be degraded to O2; the catalase concentration is high enough to avoid any oxidative stress. In this approach the chamber is not opened and closed, so the disturbance of the system is less and the stabilization phase of the sensor is short compared to a re-oxygenation by opening the chamber.<br />
<br />
*'''Re-oxygenation by opening the chamber'''<br />
<br />
:Lift the stopper and leave a gas volume above the liquid phase (use the stopper-spacer tool to set the stopper in the right position). Leave the chamber open till oxygen is again up to approximately air calibration level. Close the chamber by inserting the stopper completely and wait till the sensor is stable again (may take 5 to 10 minutes). <br />
:If possible, it is preferable to re-oxygenate in a phase of low respiratory activity, only little amounts of oxygen are consumed during the stabilization phase of the sensor after closing the chamber. <br />
If you replace the air phase above the liquid phase by pure oxygen you can increase oxygen levels above air saturation, as it is recommended for measuring mitochondrial respiratory function in muscle biopsies.<br />
<br />
== Preparation of MiR05 (MiR06) stock solution ==<br />
* Total volume of [[solution]] = 1 litre.<br />
::1) Weigh given amounts of the [[Media:MiPNet14.13 Medium-MiR06.pdf| listed chemicals]] (except BSA and lactobionic acid) and transfer to a 1000 ml glass beaker.<br />
::2) Disrupt big lumps mechanically. It is recommended to do this before adding water, because during dissolution these lumps do not disintegrate easily.<br />
::3) Add ~800 ml H<sub>2</sub>O and dissolve using a magnetic stirrer at ~30 °C<br />
::4) Add 120 ml of [[#Preparation of K-lactobionate stock solution|K-lactobionate stock solution]].<br />
::5) Adjust the pH to 7.1 with 5 M KOH at 30 °C. <br />
::6) Dissolve the BSA in a subsample of the MiR05 stock solution and add to the final MiR05 (the separate preparation of the BSA solution is recommended, since BSA produces foams that do not dissolve easily).<br />
::7) Add H<sub>2</sub>0 to a final volume of 1000 ml.<br />
::8) Check pH again and adjust if necessary with small volumes of 5 M KOH. This solution is '''MiR05'''. MiR05 can be stored at -20 °C as described for MiR06.<br />
::9) To prepare <span style="color:#2E8B57"> '''MiR06'''</span>, add 280 000 units of catalase (100 mg of catalase powder containing 2800 u/mg solid) per litre MiR05 (280 units / ml final concentration).<br />
::10) Divide into 40 ml portions in plastic vials and store at -20 °C. <br />
::11) These storage solutions of MiR06 can be used as stock [[solution]]s. A vial is warmed up above experimental temperature, avoiding foam formation during gentle shaking. Up to 16 O2k-chambers can be filled with a 40 ml portion. It is recommended to use the stock solution on a single day only, to avoid any microbial contamination of the respiration medium.<br />
<br />
::<span style="color:#2E8B57"> '''MiR06''' </span> can also be prepared by adding 5 µl of the [[#Preparation of catalase stock solution|catalase stock solution]] directly into the O2k-chamber filled with MiR05 at the start of the experiment. The final catalase concentration in the 2 ml O2k chamber is 280 u/ml.<br />
<br />
=== Preparation of K-lactobionate stock solution ===<br />
<br />
::1) weigh 35.83 g lactobionic acid into a 250 ml glass beaker<br />
::2) add 100 ml H<sub>2</sub>O and dissolve by stirring on magnetic stirrer<br />
::2) check pH (is approx. 2.0) and neutralize with 5 M KOH<br />
::4) adjust final volume to 200 ml with H<sub>2</sub>O. It is best to use a 200 ml volumetric glass flask.<br />
::5) check pH again and adjust to 7 if necessary (5 M KOH)<br />
<br />
=== Preparation of catalase stock solution ===<br />
<br />
::'''Catalase''' lypophilized powder, 2000-5000 '''units*'''/mg, Sigma C 9322, store at -20 °C<br />
<br />
::'''Stock solution:''' 112000 u/ml (dissolved in MiR05)<br />
<br />
<br />
::'''Example:''' 'Catalase lypophilized powder, 2800 units/mg solid and 3500 units/mg protein'<br />
<br />
::1) Use 'units/mg solid' for your calculations <br />
::2) Result: 40 mg catalase powder (2800 u/mg) are dissolved in 1 ml MiR05 to obtain a catalase stock solution with 112000 u/ml.<br />
::3) Titrate 5 µl of the catalase stock solution into the 2 ml chamber to achieve a final concentration of 280 IU/ml in the chamber.<br />
<br />
'''Unit definition:''' '''* Units''' of enzymatic activitiy (u) in µmol/min; assay used by Sigma Aldrich: ' ''One unit will decompose 1.0 μmol of H<sub>2</sub>O<sub>2</sub> per min at pH 7.0 at 25 °C, while the H<sub>2</sub>O<sub>2</sub> concentration falls from 10.3 to 9.2 mM, measured by the rate of decrease of A<sub>240</sub>.'' '<br />
<br />
== MiR05Cr/MiR06Cr ==<br />
* [[MiR05Cr]] = MiR05 + Creatine<br />
* [[MiR06Cr]] = MiR06 + Creatine<br />
<br />
::1) Prepare fresh by adding 3 mg/ml creatine monohydrate (Fluka 27900, 100 g) to MiR05 or MiR06.<br />
::2) Stirr gently on a magnetic stirrer.<br />
::3) Do not freeze to avoid precipitation.<br />
<br />
== Preparation of 200 mM H<sub>2</sub>O<sub>2</sub> stock solution ==<br />
<br />
'''H<sub>2</sub>O<sub>2</sub>''': Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge. See [http://www.h2o2.com/technical-library/default.aspx?pid=66&name=Safety-amp-Handling this link] for handling and safety instructions concerning hydrogen peroxide.<br />
<br />
::1) Pipette 114 µl of 17.6 M H<sub>2</sub>O<sub>2</sub> into 10 ml plastic vial.<br />
::2) Add H<sub>2</sub>O, acidify with HCl (1 mM) to pH 6, complete with H2O to a total volume of 10 ml. Maintain the pH in the stock solution acidic to minimize autoxidation.<br />
::3) Wrap plastic vial in aluminium foil (solution is light sensitive) and store at 4 °C.<br />
::4) During experiments keep the stock solution on ice.<br />
<br />
'''Titration''' of 3 µl of H<sub>2</sub>O<sub>2</sub> into the 2 ml O2k-chamber increases the concentration of O<sub>2</sub> by approx. 150 nmol/ml (150 µM).<br />
<br />
== Limitations of using MiR media ==<br />
<br />
::* MiR06 or MiR06Cr cannot be used for measurement of ROS production. Use MiR05 or MiR05Cr instead.<br />
::* The high antioxidant activity may compete with reactions on which measurement of ROS production is based.<br />
::* The intracellular milieu of kidney has a low [K<sup>+</sup>]. Kidney mitochondria are inhibited by the high [K<sup>+</sup>] of MiR05 to MiR06Cr [1].<br />
>> [[MiPMap#1._Human_and_model_organisms.2C_taxonomic_groups|MiPMap - Is this a general issue for the organ, or is it in addition also a species issue?]]<br />
<br />
# A mitochondrial respiration medium for kidney: [[Friederich-Persson 2012 Diabetologia]].<br />
<br />
== Further information ==<br />
::* [[MiPNet06.03_O2-Calibration-Solubility |Oxygen solubility in MiR06]]<br />
<br />
::* [[MitoPedia: Media for respirometry]]<br />
<br />
::* MiPNet08.05 and MiPNet10.11 are integrated in MiPNet14.13_Medium-MiR06.<br />
<br />
'''Original publication introducing MiR05:'''<br />
* [[Gnaiger 2000 Life in the Cold|Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York: pp 431-442.]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet14.14_PermeabilizedFiberPreparation&diff=149153MiPNet14.14 PermeabilizedFiberPreparation2018-01-11T08:30:01Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols contents]]Preparation of permeabilized muscle fibres for diagnosis of mitochondrial respiratory function. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/c/c0/MiPNet14.14_PermeabilizedFibrePreparation.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet14.14_PermeabilizedFibrePreparation.pdf Versions]<br />
|authors=Oroboros<br />
|year=2011-12-02<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Pesta D, Gnaiger E (2009) Preparation of permeabilized muscle fibres for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 14.14.''' <br />
<br />
Application of [[permeabilized muscle fibres]] and [[high-resolution respirometry]] offer a sensitive diagnostic test of mitochondrial dysfunction in small [[biopsy]] specimens of human muscle. By using these techniques in conjunction with multiple [[substrate-uncoupler-inhibitor titration]] (SUIT) protocols, respirometric studies of human and animal tissue biopsies improve our fundamental understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial myopathies.<br />
<br />
[[Image:MiPNet14.14.jpg|right|200px|thumb]]<br />
:>> Product: [[O2k-Catalogue: O2k-MultiSensor]], [[O2k-Core]], [[Oroboros O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Human<br />
|tissues=Heart, Skeletal muscle<br />
|preparations=Permeabilized tissue<br />
|instruments=O2k-Protocol<br />
|additional=Mt-Preparation<br />
}}<br />
== Further Information ==<br />
'''References'''<br />
<br />
* [[Pesta_2012_Methods_Mol_Biol| Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.]]<br />
<br />
* [[Lemieux_2011_Int_J_Biochem_Cell_Biol| Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43: 1729–1738.]]<br />
<br />
* [[Votion_2012_PLoS_One| Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7(4): e34890.]]<br />
<br />
<br />
=== Preparation of permeabilized muscle fibres ===<br />
<br />
==== Requirements ====<br />
<br />
Ketamine, xylazine; 2 pairs of sharp scissors (large and small); 2 pairs of forceps with sharp tips (one straight tip, one angular tip; non-magnetic), 2 pairs of forceps with very sharp angular tips (non-magnetic); ice; tissue culture plate with 12 wells (Falcon 35/3043); 5 ml beaker; magnetic stirrer plate; small (3 ml) test tubes, microbalance (5 digits; 0.01 mg).<br />
<br />
BIOPS: The relaxing and biopsy preservation solution BIOPS contains 10 mM Ca-EGTA buffer, 0.1 µM free calcium, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM DTT, 6.56 mM MgCl2, 5.77 mM ATP, 15 mM phosphocreatine, pH 7.1 [MiPNet03.02]. BIOPS can be stored and shipped frozen at -20 °C.<br />
<br />
Animals: After full anaesthesia of the the mouse or rat through intraperitoneal injection (e.g. Fentanyl, 1 ml/kg of mouse), the condition of the animal is checked (heart beat is visible, the reflex test on the hindlimb is negative).<br />
<br />
==== Tissue preparation ====<br />
<br />
The muscle (heart or skeletal muscle) is excised. Skeletal muscle is cut lengthwise into small samples of 10-20 mg Ww, and put into a Falcon tube with 10 ml of ice cold BIOPS. In this preservation solution, the sample can be stored for several hours at 0 °C, depending on the source of muscle tissue (e.g. mouse skeletal muscle for at least six hours; human skeletal muscle for up to 24 h; Skladal et al 1994). This provides the possibility for shipping of biopsies on ice for functional analysis by high-resolution respirometry. <br />
<br />
Place the tissue sample in ice cold preservation solution (BIOPS) into a small petri dish on ice. Remove all connecting tissue using the pair of sharp or very sharp forceps.<br />
<br />
==== Tissue separation ====<br />
<br />
Fibre bundles are separated mechanically with the two pairs of very sharp forceps, in a small petri dish on ice. A period of five minutes is required for preparation of a 2-mg sample of fibres. The degree of separation may be evaluated by observing a change from red to pale colouring of the separated fibre bundles. At least during an initial start-up period, use of a dissecting scope is recommended for observation of the mechanical separation, during which fibres are partially teased appart and streched out. The tissue should still remain connected in a mesh-like framework. <br />
<br />
This preparation leads to partial permeabilization of skeletal muscle, but full permeabilization of cell membranes in heart (Kuznetsov et al. 2004) or liver tissue (Kuznetsov et al. 2002). <br />
<br />
After tissue separation, the fibre bundels are placed sequentially into 2 ml ice cold BIOPS into individual wells of a Falcon 12-well tissue culture plate. <br />
<br />
==== Tissue permeabilization ====<br />
<br />
After completion of separation of fibre bundles for two or more O2k-chambers, the fibre bundles are transferred quickly into 2 ml of icecold BIOPS, containing 20 µl of saponin stock solution (5 mg/ml; final concentration 50 µg/ml). <br />
<br />
Shake by gentle agitation in the cold room (on ice) for 30 min. After the 30 min period, all samples are quickly transferred from the saponin solution into 2 ml of MiR06 (see figure), and shaken by gentle agitation for 10 min in the cold room (on ice).<br />
<br />
Additional samples may prepared with or without saponin permeabilization for other assays (histochemical/morphological; enzymatic; mtDNA). <br />
<br />
=== Wet weight ===<br />
<br />
Weight measurements are made after permeabilization, eliminating variations in water contents due to osmotic stress, allowing for elimination of connective tissue during mechanical separation before weight measurement, and for partitioning of subsamples of similar wet weight.<br />
<br />
Before adding the tissue into the O2k chamber, take wet weight measurements of several loosly connected fibre bundles of c. 1-3 mg wet weight (skeletal muscle) or 0.5-2 mg wet weight (heart). Blot the bundles carefully on filter paper. Take the sample with the sharp pair of forceps (angular tip) and place it for 5 s onto the filter paper. During this time, wipe off any liquid from the tip of the forceps with another filter paper. Then take the sample from the filter paper and touch it once more shortly onto a dry area of filter paper while holding it with the forceps. Then immediately place the sample onto a small plastic plate on the table of the tared balance.<br />
<br />
Immediately after reading the wet weight, the sample is transferred into a well with 2 ml ice cold MiR06. Check that the tare balance reading returns to zero. Each well receives a sample for an O2k-chamber.<br />
<br />
=== Oxygraph-2k ===<br />
<br />
Calibrate the oxygen sensors in MiR06 at experimental temperature in the ‘open’ chamber (gas phase above the stirred medium, with a partially inserted stopper) of the Oxygraph-2k at a Gain of 1, to ensure that the raw signal does not reach the limiting value of 10 V at high experimental oxygen concentrations.<br />
<br />
Respiration is measured (at 37 °C for mammalian tissues) in mitochondrial respiration medium (MiR06) designed for optimal protection of mitochondrial function [MiPNet14.13]. From the vial with MiR06, the sample is transferred into the Oxygraph-2k chamber containing air saturated MiR06. The sample is immersed with the pair of straight forceps into the stirred medium in the O2k-chamber.<br />
<br />
After closing the chamber, the oxygen concentration is increased to 350 µM, adding microliter volumes of H2O2 solution, using the TIP2k or by manual titration [MiPNet14.06]. The oxygen signal increases rapidly at each titration step, and the titration is continued until the final oxygen level is reached. 5 to 10 min are required for stabilization of the signal. Oxygen concentration is prevented to drop below 200 µM by intermittent H2O2 titrations, preventing diffusion limitation of respiration in permeabilized fibres (Gnaiger 2003).<br />
<br />
Respiratory flux of muscle fibre bundles is limited by oxygen diffusion even above 50% air saturation (O2 concentration of 100 µM). This problem is carefully controlled and avoided by application of a high-oxygen regime. It is essential that instrumental and chemical oxygen background fluxes are routinely determined as a function of oxygen concentration in the experimental range. With appropriately calibrated background parameters inserted into the ‘Edit Experiment’ window of Datlab 4, background corrections are automatically obtained on-line over the entire experimental oxygen range. This is an indispensable prerequisite for accurate measurements particularly at low fluxes, when the amount of biological material is limited.<br />
<br />
=== References ===<br />
<br />
# [[Gnaiger_2003_Adv Exp Med Biol|Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55.]]<br />
# [[Gnaiger_2000_MitoInTheCold|Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heiderlberg, Berlin, New York: 431-442.]]<br />
# Lemieux H, Gnaiger E (2007-2008) Preparation of permeabilized muscle fibers for diagnosis of mitochondrial respiratory function. MiPNet12.22: 1-4.<br />
# [[Pesta 2012 Methods Mol Biol|Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.]]<br />
# Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS (1998) Permeabilised cell and skinned fibre techniques in studies of mitochondrial function in vivo. Mol Cell Biochem 184: 81-100.<br />
# [[Skladal_1994_BTK|Skladal D, Sperl W, Schranzhofer R, Krismer M, Gnaiger E, Margreiter R, Gellerich FN (1994) Preservation of mitochondrial functions in human skeletal muscle during storage in high energy preservation solution (HEPS). In: What is Controlling Life? (Gnaiger E, Gellerich FN, Wyss M, eds) Modern Trends in BioThermoKinetics 3, Innsbruck Univ Press: 268-271.]]<br />
# Kuznetsov AV, Gnaiger E (1998) MiPNet03.01 and (1999) MiPNet04.07.<br />
# [[Kuznetsov_2004_Am J Physiol Heart Circ Physiol|Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286: H1633–H1641.]]<br />
<br />
Protocols:<br />
<br />
* [[MiPNet03.02]] Selected media and chemicals.<br />
* [[MiPNet14.06 Instrumental O2 background]] correction and accuracy of oxygen flux.<br />
* [[MiPNet14.13]] Mitochondrial respiration medium – MiR06.</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet15.03_O2k-MultiSensor-ISE&diff=149146MiPNet15.03 O2k-MultiSensor-ISE2018-01-11T08:18:28Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-MultiSensor system with ion selective electrodes (ISE).<br />
|info=[[Image:PDF.jpg|100px|link=http://wiki.oroboros.at/images/a/a8/MiPNet15.03_MultiSensor-ISE.pdf |Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:MiPNet15.03_MultiSensor-ISE.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-03-06<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M, Gnaiger E (2014) O2k-MultiSensor system with ion selective electrodes (ISE). Mitochondr Physiol Network 15.03(05):1-20.''' - <br />
<br />
'''O2k-Manual''': The ion selective electrode (ISE) is a modular O2k-MultiSensor extension of the Oroboros O2k. ISE yield a potentiometric (voltage) signal simultaneously with the oxygen signal in both chambers of the O2k. The ISE system consists of separate reference and measuring electrodes selective for hydrophobic cations (TPP<sup>+</sup>, TPMP<sup>+</sup>), Ca<sup>2+</sup>, Mg<sup>2+</sup>, etc. This O2k-Manual describes the handling and application of the ISE system.<br />
:» Product: [[O2k-TPP+ ISE-Module]], [[Oroboros O2k-Catalogue]]<br />
|keywords=[[HRR]], [[O2k-MultiSensor]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=Oxygraph-2k, TPP, O2k-Manual, O2k-Protocol<br />
|additional=O2k-MultiSensor, DatLab, DL6, DL7, DL6a7<br />
}}<br />
<br />
{{Technical support integrated}}<br />
<br />
<br />
__TOC__<br />
<br />
== High-resolution respirometry and mt-membrane potential ==<br />
<br />
:::: The O2k-MultiSensor system provides a potentiometric and a fluorometric module for measurement of the mt-membrane potential.<br />
<br />
:::: The [[TPP+ inhibitory effect]] on respiration should be explored when applying TPP, and unspecific binding should be considered when calculating the mt-membrane potential: [[MiPNet15.08 TPP electrode]].<br />
<br />
<br />
=== O2k signal and output ===<br />
::::# [[O2k signals and output#Signal of the O2k-Core and add-on modules |O2k signal]]: The [[O2k-TPP+ ISE-Module]] is operated through the pX channel of the O2k, with electric potential (volt [V]) as the primary and raw signal<br />
::::# [[O2k signals and output#O2k output |O2k output]]: type C<br />
<br />
:::: In Series D and higher the ISE connectors consist of 2 ports:<br />
::::# A BNC port: both poles are "live" so it can be used either for a combination electrode (like a classical pH) or for just one singe electrode that requires a separate reference electrode.<br />
::::# A 2 mm pin type connection that is used for connecting the separate reference electrodes for TPP and pH.<br />
<br />
=== Standard cleaning procedure for TPP+ and reference electrodes ===<br />
::::The PVC membranes of the ISE are generally only suitable for operation in aqueous media and are damaged by non-aqueous solvents. Therefore, the necessary washing steps between experiments have to be carefully optimized according to specific experimental regimes.<br />
<br />
::::# Rinse the electrodes after use with water, dry with paper, rinse with absolute ethanol, again dry with paper and rinse with water again.<br />
::::# Store in a separate falcon tube with a proper storage solution.<br />
::::# When inhibitors and uncouplers have been used, continue by putting the electrodes into a falcon tube filled with distiled water for 2-5 min. Then switch to the falcon tube with a warm (30-37°C)'biological cleaning sample' for 20-30 min. Liver homogenate, isolated mitochondria or cell cultures can be used as biological cleaning sample, which can be stored at -20°C.<br />
::::# Alternatively, the electrodes can be washed with biological cleaning sample added to the O2k-chamber, cleaning the insterted electrodes and the O2k-chamber together.<br />
::::# If a carry-over of inhibitors is observed despite of the above cleaning steps, immerse the TPP electrode in absolute ethanol for a few minutes in the O2k-chamber, with stirring. However, afterwards the electrodes have to be tested and a reduced lifetime for the membranes is to be expected.<br />
::::# [[Talk:MiPNet15.03 O2k-MultiSensor-ISE |Discussion]]<br />
<br />
=== TPP electrode - interaction with TMPD ===<br />
::::* TMPD traveling into the TPP probe: pretty sure it was happening because the electrolyte was turning blue. And actually it became a persistent problem with several probes (at first I thought maybe there was a leak around the membrane off of one probe) (it would shift the absolute voltage values to more positive, until it the signal gets super staticky and I couldn't use the data at all). I resorted to either making sure I inverted the probe a few times to redistribute the TMPD in the electrolyte to spread it out a bit... or switching out the electrolyte (without replacing the membrane) and letting it sit overnight. And of course minimizing contact time of the probe with a solution containing TMPD.<br />
:::: ~ Contribution by [[Lau G|Gigi Lau]] from [[CA Vancouver Richards JG]].<br />
<br />
=== Long-term storage of the TPP electrode ===<br />
:::: For long-term storage (several months) disassemble the electrode, wash all parts with ethanol (sterilization), and allow all parts to dry completely. Then re-assemble the dry electrode without a membrane (avoid loosing the small parts). Store the electrode in the TPP accessory box (dark).<br />
<br />
<br />
=== Mitochondrial membrane potential and anoxia ===<br />
<br />
:::: Can anoxia be used as a reference state for zero (minimum) mt-membrane potential in isolated mitochondria? This might save the time for washing out inhibitors or uncouplers. The protocol includes substrates and ADP.<br />
::::# Anoxia should provide a good reference value for minimum mt-membrane potential. However, you should carry out a test experiment: After reaching anoxia, add oligomycin as a test for the possibility that ATPsynthase acts as a ATPase and thus maintains a mt-membrane potential in reversed mode of operation. Then titrate uncoupler (FCCP) to collaps the mt-membrane potential under anoxia.<br />
::::# Careful: Ethanol as a carrier for oligomycin and FCCP exerts a chemical side effect on the TPP+ signal, which has to be evaluated in a separate control experiment and subtracted from the experimental trace.<br />
<br />
<br />
== Correction for substance-specific effects on the TPP signal ==<br />
<br />
:::: The necessity to perform a TPP chemical background experiment is explained in MiPNet 14.05. Some additional considerations:<br />
<br />
=== When to apply a correction ===<br />
:::: For isolated mitochondria absolute delta delta Psi values seem obtainable, see above. Approximate delta Psi values seem to be principally obtainable, though with relying on literature data. The strongly quantitative approach enabled thereby calls for complete quantifications including correction for unspecific effects.<br />
:::: For permeabilized cells, homogenates, and permeabilized fibres, absolute values of delta or delta delta Psi seem currently difficult to obtain. Data will have to presented as a relative value. Therefore, a discussion about to apply or not to apply a correction for substance specific effects seems justified: Whenever changes of mitochondrial membrane potential during an experiment are of interest a correction is most definitely needed. Otherwise even the nature of the change (increase / decrease) may be misjudged.<br />
:::: When membrane potentials obtained by different protocols but using the same parameters (binding correction factors) during calculation should be compared to each other, correction for substance specific effects has to be done, even though only relative values are compared to each other.<br />
:::: When relative values for membrane potentials of the same state obtained via totally identical protocols are to be compared between different samples a correction may not be strictly necessary. In this case the research will have to judge on a case basis. If the correction is obviously rather difficult, the danger of introducing additional errors may be greater than any benefit from getting slightly more realistic values.<br />
:::: Even if it is decided for a particular study not to apply the correction the TPP+ chemical background experiments should be done non the less to detect possible problems.<br />
<br />
=== Substances ===<br />
:::: Azide, N3-, has a very huge substance-specific effect. A correction does not seem feasible.<br />
::::The substance-specific effect of ADP is comparably large and should be considered carefully.<br />
<br />
<br />
== Mitochondrial membrane potential of permeabilized fibres ==<br />
<br />
:::: Based on a report by [[Lin CT|Lin Chien-Te (Peter)]], [[US NC Greenville Neufer PD|Darrell Neufer at East Carolina University, Greenville, NC, USA]] and contributions by Mario Fasching and [[Sumbalova Z|Zuzana Sumbalova]].<br />
<br />
=== General ===<br />
<br />
:::: From experiments with isolated mitochondria or permeabilized cells one can derive the concentration and mitochondrial activity (oxygen flux per volume) necessary to obtain reliable signals with the TPP<sup>+</sup> electrode. Since unspecific binding is higher om [[Permeabilized muscle fibre|permeabilized muscle fibers]] compared to isolated mitochondria, the amount of fibres should be chosen to obtained rather high volume-specific oxygen fluxes. Even with permeabilized cells higher sample concentrations are required, as compared with standard high-resolution respiratory measurements. It is important that the total amount of TPP in the chamber is known at all times. Therefore, the sample should not be preconditioned outside of the chamber to TPP, and even a rough estimation of the sample volume will be necessary.<br />
<br />
=== Introduction of the sample ===<br />
<br />
:::: The established way to measure mitochondrial membrane potential for isolated mitochondria (and permeabilized cells) is to calibrate the TPP electrode by adding TPP in several steps to the experimental chamber. With the final calibration step the starting TPP<big>+</big> concentration is reached. Then the sample is injected into the "calibrated" chamber. Therefore, unlike in the application of other potentiometric methods (pH, Ca<sup>2+</sup>,..) the "calibration" does in fact serve two different purposes:<br />
::::# Calibration of the sensor;<br />
::::# Establishing the total amount of TPP in the chamber. This amount has to be precisely known for calculation of mtMP from the measured [TPP<sup>+</sup>].<br />
<br />
:::: Introduction of the sample is a key problem. Removing the stoppers and placing the permeabilized fiber into the chamber results in a disturbance of the TPP electrode calibration. It would be necessary to replace all medium lost with medium containing exactly the TPP concentration established in the O2k-chamber before opening it. However, after introducing permeabilized fibers they immediately start to take up TPP. Opening and closing the chamber typically requires quite a lot of “bubble fighting”.<br />
<br />
::::* The recommended approach is:<br />
::::# The TPP electrode is calibrated up to 1. 0 or 1.5 µM [TPP+] at a high O2 level of >500 nmol/ml.<br />
::::# Lift the stopper with electrodes just slightly (stirrer off), such that the TPP electrode is still immersed in the Medium. <br />
::::# Remove the reference electrode.<br />
::::# Introduce fibers through reference electrode port using a glass Pasteur pipette cut to the length 16.2 cm. The cut edge was smoothed in a flame. The fibres are taken up into the pipette and gently introduced into the chamber. <br />
::::# The wet weight is about 3 mg per chamber for mouse cardiac muscle.<br />
::::# Close the chamber without bubbles and switch on the stirrers.<br />
::::# Due to the high O2 flux frequent reoxygenations are required.<br />
<br />
:::: The disturbance of the calibration by removing and re-inserting the reference electrode is minimal. Removal and reinsertion of the reference electrode should be done with stirrers switched off. The fibre bundles are split into several parts if necessary.<br />
<br />
<br />
=== Reoxygenation and high oxygen ===<br />
<br />
:::: The method recommended by Oroboros to do a re-oxygenation in the presence of additional electrodes is to inject H2O2 into a medium containing catalase, avoiding any mechanical disturbances, see the protocol for the MiR06 medium [[MiPNet14.13]]. Because the H2O2 method is limited to a delta cO2 of 200 µmol/l the initial high oxygen concentration should be achieved with high oxygen in the gas pahse before starting the experiment. The O2 level can then be maintained by H2O2 injections without further opening the chamber.<br />
<br />
=== Slowness of TPP uptake and release ===<br />
:::: TPP uptake and release seems generally to be slower for permeabilzed fibers than for isolated mitochondria or permeabilized cells. However, the extent of this effect was reported to be very different by different groups. It is not yet clear what causes extremely slow uptake/ release in some cases but not in others.<br />
<br />
<br />
== Calculation of mitochondrial membrane potential from measurement of TPP<sup>+</sup> ==<br />
<br />
:::: Based on information provided in the O2k-Protocols [[MiPNet14.05 TPP-mtMembranePotential |MiPNet 14.05]], which should be consulted first. The most up to date spreadsheet templates, DatLab templates, DatLab demo files, MiPNet14.05 and its mathematical appendix can be found [[MiPNet14.05 TPP-mtMembranePotential|here]].<br />
<br />
:::: The calculation of mitochondrial membrane potential from measurements with a TPP electrode is a difficult and far from settled topic.<br />
<br />
<br />
=== Sensitivity analysis of the method ===<br />
[[File:Error_evaluation_absolute_1pc.png|thumb|300px|alt=absolute error in delta Psi by introduction of a 1 % error in c(TPP) plotted against delta Psi|absolute error in delta Psi by introduction of a 1 % error in c(TPP) plotted against delta Psi]]<br />
<br />
:::: The sensitivity of the method to small errors is strongly dependent on the membrane potential. For low membrane potential the method is inherently unsuitable. This is illustrated by introducing an artificial errors in the measured [[TPP]]+ concentration and plotting the resulting errors in the calculated membrane potential against the (original) membrane potential. The exact shape of the function depends on sample amount and type, binding correction and all other external factors but the general shape is usually quite constant.<br />
:::: Here this is illustrated for isolated (un-purified) mitochondria, simulating the effect of a +1% and -1% error in the measured TPP+ concentration. <br />
<br />
:::: The used calculation template is not able to deal properly with results that would lead to a negative membrane potential, therefore errors leading to a 0 or negative membrane potential are shown here as "zero".<br />
<br />
=== Unspecific binding ===<br />
<br />
==== The four compartment model ====<br />
:::: The approach to unspecific binding chosen in MiPNet 14.05 and in the Oroboros Spreadsheet temples is basically based on Rottenberg's <ref name ="Rottenberg1984">Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81:127-38.</ref> 4 compartment model, developed for isolated mitochondria. As shown in the mathematical appendix to [[MiPNet14.05 TPP-mtMembranePotential|MiPNet 14.05]] this approach seems to be mathematically fundamentally equivalent to the approaches by Brand <ref name="Brand1995">Brand MD (1995) Measurement of mitochondrial protonmotive force. In: Bioenergetics a practical approach (Brown GC, Cooper CE, eds):39-62. Oxford University Press, Oxford.</ref> and Kamo <ref name="Demura1987">Demura M, Kamo N, Kobatake Y (1987) Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis. Biochim Biophys Acta 903:303-8.</ref>, at least for the processes inside the mitochondria.<br />
:::: Four compartments are considered:<br />
<br />
::::# The liquid filled matrix of the mitochondria, containing “free, internal” TPP+. <br />
::::# Material (membranes etc ) exposed to the typically high TPP+ concentration in compartment A. In Rottenbergs original approach this is the inside face of the inner mitochondrial membrane.<br />
::::# The liquid filled space outside the mitochondria. This comprises the entire volume of the sample chamber with the exception of compartments A, B, and D.<br />
::::# Material (membranes etc) that are exposed to the typically low TPP+ concentrations outside the mitochondrial matrix. In Rottenberg's original approach this compartment comprises the outside face of the inner mitochondrial membrane and any present material from the outer mitochondrial membrane or traces of cell material not removed during purification.<br />
<br />
:::: The probe ion is supposed to accumulate in compartments B and D directly proportional to <br />
::::* the “size/amount” of the compartment, measured by some marker, e.g. protein content<br />
::::* the concentration of probe molecule in the adjunct liquid phase, e.g the TPP+ concentration in the mitochondrial matrix<br />
::::* a factor describing the affinity of the compartment to the probe molecule (the binding correction factor.<br />
<br />
:::: E.g. the amount of TPP+ "bound" by the inward facing side of the inner mitochondrial membrane is<br />
<br />
:::: ''n''(int,bound) = ''K''i' * Pmt * ''C''(int,free)<br />
<br />
:::: (Equation A8a in the mathematical Appendix to MiPNet14.05)<br />
<br />
:::: A binding correction factor (e.g. ''K''i’) is only useful together with a certain type of marker (Pmt) for which it was determined.<br />
<br />
:::: The approaches by Brand and Kamo do not consider the outside compartments for unspecific binding. Indeed, for purified isolated mitochondria the outside binding seems to have a very small effect. Therefore in all further considerations one has to discern between studies of purified isolated mitochondria and studies with other sample types.<br />
<br />
=== Isolated mitochondria and unspecific binding in the mitochondria ===<br />
:::: Due to the small amount of material exposed to the outside concentrations and the low outside concentrations only the inside binding is significant.<br />
:::: The absolute values for delta Psi will depend on the chosen binding correction factors.<br />
<br />
:::: The absolute DIFFERENCE between membrane potentials (either between different states or different samples) will NOT depend on the chosen inside binding correction factor, see MiPNet 14.05 Mathematical Appendix. Therefore, it should be possible to obtain absolute change of delta Psi’s for this sample type. The insensitivity against outside binging can be shown by varying the outside binding parameter only:<br />
<br />
[[File:Isolated mito Kout variation.png|500px|alt=various delta PSi values and one delta delta Psi values plotted against varying external binding parameter Kout'|various delta PSi values and one delta delta Psi values plotted against varying external binding parameter Kout']]<br />
<br />
:::: Only a few binding correction factors for internal binding have been published, based on rat liver mitochondria or membrane models under very different conditions (temperatures, mitochondrial membrane potential,…) While different mathematical approaches were used to describe the binding an attempt to convert these factors between different mathematical models shows quite similar values for the probe TPMP+ (the probe for which most values are available):<br />
<br />
::::* Brand<ref name="Brand1995"/>: 2-3.2 µl/mg (converted to Rottenberg's system)<br />
::::* Rottenberg<ref name ="Rottenberg1984"/>: 2.4 to 3.7 µl/mg<br />
::::* Kamo<ref name="Demura1987"/>: 2.7 µl/mg (converted to Rottenberg's system)<br />
<br />
:::: Simultaneous variation of outside and inside binding parameters show: <br />
::::* the invariance of delta delta Psi<br />
::::* that strong deviation from published Kin' values do not lead to reasonable results:<br />
[[File:Isolated mito KinandKout variation.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external and internal binding parameter Kout'|various delta Psi values and one delta delta Psi values plotted against varying external = internal binding parameter Kout']]<br />
<br />
<br />
:::: Conclusions for isolated isolated mitochondria:<br />
::::* Absolute change of delta Psi values can be obtained.<br />
::::* Precise absolute delta Psi values can not be obtained without actually measuring the binding correction factor for the studied system. Literature values will usually not be available for the desired system. Approximate delta Psi values may be obtainable by using literature values, if variances in "unspecific binding" between sample types and conditions are small (still to be shown).<br />
<br />
=== Permeabilized cells, homogenates, permeabilized fibres and unspecific binding outside the mitochondria ===<br />
<br />
:::: In these sample types the determination of mitochondrial protein present is more complicated than for isolated mitochondria. Estimations may be based on the observed O2 flux, or on using a other marker for the presence of mitochondrial activity (citrate synthase). If the amount of mitochochondrial protein was estimated wrongly this may lead do drastically and obviously wrong absolute membrane membrane potentials.<br />
:::: Below the influence of different assumption for the amount of mitochondrial protein in a preparation of brain homogenate. Delta delta Psi values between states of reasonable high membrane potential are not affected.<br />
<br />
[[File:Homogenate brain centrifuged Pmt.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying amount of mitochondrial protein Pmt'| brain homogenate, centrifuged: various delta Psi values and one delta delta Psi values plotted against varying amount of mitochondrial protein Pmt']]<br />
<br />
:::: In these sample types there is a large amount of materials outside the mitochondrial matrix present. But potentially even more difficult than the absolute amount of material is the variety of materials. Inside the mitochondrial matrix the mitochondrial membrane is the only type of material taking up the probe ion and can therefore be accurately described by a single binding correction factor. Outside the mitochondria there may be membranes, proteins, other lipid compartments and even components of the medium to consider. It is reasonable to expect that all of them show a different affinity to TPP+ or other probe ions. <br />
<br />
:::: In theory, the four compartment approach can be applied to such samples. All outside material will be exposed to the low extra-mitochondrial probe ion concentration and can therefore be included in compartment D. Due to the different nature of the outside material it can be expected that a quite different binding correction factor will be needed than the one determined by Rottenberg for the outside binding to isolated mitochondria. Additionally, it may be discussed what would be a good marker for the amount of outside material present. It should be remembered that each binding correction factor is only valid for the use with a specific marker quantity (like protein content).<br />
:::: From a mathematical point of few the contribution of outside binding does not cancel even for the determination of change of delta Psi.<br />
:::: However, the first question before addressing this problems is whether outside binding is relevant at all. Brand<ref name="Brand1995"/> stated that for permeabilized cells outside binding may be ignored for high mitochondrial membrane potential. Initially, this seemed to be confirmed by our own initial sensitivity studies. Using outside binding correction factors similar to the inside ones and using protein content as marker, changing the outside binding correction factor by several hundred percent caused comparable small changes in reasonable high membrane potentials and negligible changes in delta delta Psi values for permeabilized cells. However, with growing experience it became evident that unspecific binding may be underestimated by this approach, resulting in obviously too high membrane potentials especially for states of known low potential. Part of the unreasonable high membrane potential could be explained by wrong assumptions for the amount of mitochondrial protein (Pmt). Non the less, modeling of the outside binding correction factor showed that sometimes the correction had to be increased by factors above 25 to model reasonable membrane potential. With such a huge contribution of outside binding also differences between states (delta delta Psi) are now very significantly influenced by the choice of the outside binding correction factor. A bit surprisingly, very high membrane potentials still change only very little even when the outside binding correction factor is changed by more than a factor of 25.<br />
<br />
[[File:Homogenate brain centrifuged Kout.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'| brain homogenate, centrifuged: various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]<br />
<br />
:::: One problem with this approach, at least in the shown example, may be that medium membrane potential values (e.g. ADP) decrease quite strongly with increasing external binding, resulting in a very strong increase in differences (changes of delta Psi), even if the low potential states are modeled not to a delta Psi of zero but one similar to the values observed for isolated mitochondria.<br />
:::: However, in other but similar experiments medium high membrane potentials (ADP) and changes of delta Psi were more stable against variation of ''K''out'.<br />
<br />
[[File:Homogenate brain crude Kout.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'|crude brain homogenate: various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]<br />
<br />
:::: More comparative values both from isolated mitochondria and from homogenate/ permeabilized fibers of the same sample type would be necessary to evaluate this strategy.<br />
:::: The statement that outside binding may be ignored in permeabilized cells for high membrane potentials was actually verified, only with the restriction that this hold only true for the very highest membrane potentials obtainable. This is potentially an important observation for researchers more interested in comparing just one state between different samples. It might even be argued that for very high membrane potentials an absolute delta Psi may be estimated regardless of the used binding parameters. However, this certainly needs further evaluation.<br />
:::: By increasing both the interior and external binding parameters it is again (as with isolated mitochondria)seen that strong deviation from published Kin' values do not lead to reasonable results: <br />
<br />
[[File:Homogenate brain centrifuged Koutand Kin.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'|various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]<br />
<br />
:::: There are currently no good methods known to determine the outside binding with the possible exception of radio-tracer experiments similar to those used to determine inside binding. Even if such experiments were done, due the heterogeneity and diversity of materials found in the outside compartment, the results would be less transferable to other sample types than the results for inside binding. An obvious way out would be to use a known state of zero membrane potential to determine either all or at least just the outside binding correction factor. This approach faces two problems:<br />
::::# It is not clear how a state of reliable zero membrane potential can be reached. The true membrane potential at “low potential” states, like after addition of FCCP, may or may not be zero.<br />
::::# As discussed above the accuracy of the entire method inherently decreases with decreasing membrane potential. At zero membrane potential the smallest error in measured TPP+ concentration will cause huge errors in delta Psi. In effect the point of lowest accuracy would be used to calibrate the entire method.<br />
<br />
:::: However, at least to obtain a plausibility analysis it is certainly helpful to look at this low membrane potential states. A thorough literature search for membrane potentials obtained(with a radio-tracer method) e.g. after treatment with FCCP should be performed. Maybe an solution would be to use literature values obtained for unspecific binding in isolated mitochondria to model the inside binding but use a (crude) approximation of outside binding by observation of a zero mitochondrial membrane potential state.<br />
<br />
:::: In summary, there are two obvious ways to obtain binding correction parameters that will allow a more quantitative approach:<br />
::::* direct determination of outside binding,<br />
::::* comparison with results obtained for isolated mitochondria under as similar as possible conditions followed by fitting the binding parameters to obtain comparable results for both types of sample preparation.<br />
<br />
:::: Both approaches face several theoretical and practical differences, but should be further explored.<br />
<br />
=== Further modeling options ===<br />
:::# Saturated binding: The four compartment model could be extended by further parameters. One possibility would be to allow for a saturable component of "binding". The amount of TPP+ bound would depend only on some proportionality factor and the amount of biological material present but not on the free TPP+ concentration near the compartment. Such a behavior could be detected by performing experiments at different TPP+ levels. To obtain significant differences it would be probable necessary to use very different TPP+ concentrations (Factor 10) resulting in inhibition by TPP+ for the higher concentration studied. This might be solved by using only results at low membrane potentials. However, at low membrane potentials the accuracy of the method is inherently low.<br />
:::# It was suggested to use the quantity "taken up TPP+ per mass of sample (protein content)" as a relative expression for the membrane potential for given experimental conditions. One advantage is that if the result is to be a relative number anyway, it may be easier to argue (e.g. with reviewers) to use this expression than to calculate some delta Psi and than declaring: "This is not really delta Psi, but some relative value". On the downside, the comparability between different experimental conditions is certainly worse than with some calculated "relative delta Psi, plus even for the same experimental conditions the relationship between the stated number and true membrane potential (especially the linearity of the relationship!) may be worse. This should be checked by calculations / simulations.<br />
<br />
:::: While probable not utilizing the measured data to its full extent this approach might be quite a safe way to present some minimum information of the data.<br />
:::# Methods based on different kinetics of unspecific binding vs mt uptake.<br />
<br />
<br />
== Simultaneous measurement of TPP and fluorescence ==<br />
:::: A black TPP stopper is required. Light entering the chamber through the TPP electrode might be a problem. However, producing the TPP electrodes in black is presumable not a very good idea because then it will no longer be possible to "see" the position of the membrane. In preliminary tests, a chamber was equipped with TPP electrodes (black stoppers) and a fluorescence module for measuring H2O2 via the Amplex Red method. A standard TPP calibration was carried out.<br />
:::: Amplex red / HRP addition resulted only in a minor signal in the TPP channel, a further TPP calibration showed no obvious large negative effects on the performance of the TPP electrode. A H2O2 calibration of the fluorescence signal was carried out: The '''sensitivity''' for H2O2 was reduced by a '''factor of 3 to 4''' as compared to measurements without TPP electrodes. At the same time the noise was drastically increased. In the comparative experiment (without TPP electrodes) no random noise was seen - digital resolution was the limiting factor. Based on this comparison, '''noise''' increased in the experiment with TPP electrodes at least by a '''factor of 20''', probable more. It was further tested whether the presence of TPP+ alone (without electrodes) could explain this behavior but, if there were any effects at all, they were quite small. Peroxide additions corresponding to a concentration change of 110 nM (110 pmol/ml) were still well visible but not additions corresponding to a 22 nM (22 pmol/ml) concentration change. Parallel measurement e.g one chamber TPP / one chamber H2O2 are preferable. <br />
<br />
:::: The O2k-system is very flexible. If you take cultured cells or isolated mitochondria, split them into the two chambers of the O2k, then you can maximize the number of simultaneously measured parameters: <br />
::::* In both chambers oxygen concentration and oxygen flux ([[O2k-Core]]);<br />
::::* In both chambers fluorescence, with either identical fluorophores and identical optical probes, or different ones for different parameters in each chamber (H2O2, ATP production, Ca2+, mt-membrane potential, with the potential to extend these possibilities; [[O2k-Fluo LED2-Module]]).<br />
<br />
:::: Depending on fluorophore compatibility, additionally one potentiometric electrode can be inserted into each chamber (pH, TPP+, Ca2+; [[O2k-TPP%2B_ISE-Module]]);<br />
<br />
:::: Or in one chamber fluorescence and the other chamber NO ([[O2k-NO_Amp-Module]]).<br />
<br />
:::: Importantly, oxygen concentration is not only measured, but oxygen levels can also be controlled. This marks a new dimension in our evaluation of ROS production, with measurements spanning the entire ‘normoxic’, hyperoxic and deep hypoxic range.<br />
<br />
<br />
== Technical service for the ISE system ==<br />
:::: Sometimes it is difficult to find out whether problems with the ISE system are caused by the electrodes (TPP, Ca, reference, pH) or by the pX electronics of the O2k. The following tests can help to solve this question.<br />
<br />
=== Testing a TPP system outside the O2k-Chamber ===<br />
:::: This test allows to test for any disturbing influences in the chamber, like crosstalk to the [[OroboPOS]]. It is therefore similar to the test proposed in the manual (switching off the O2 polarization voltages) but has a broader scope (covering also e.g. possible interference from stirrers) and provides a more complete separation of POS an pX electrodes.<br />
<br />
:::: Place TPP electrode and reference electrode together in one falcon tube filled with electrolyte solution (e.g. media). Connect the cables of the electrodes to the O2k and record the pX signal. If a problem previously observed disappears in this test (and is reproducible by using the same electrodes in the O2k-Chamber) the problem is located in the chamber, e.g. at a leaky POS membrane, see the manual. <br />
<br />
<br />
=== Testing a TPP system with a pH electrode (and vice versa) ===<br />
:::: This method allows to test the sensitivity of the pX electronics.<br />
<br />
::: '''Requirements'''<br />
<br />
:::: Another type of electrode than can be connected to the ISE system. So if the problem is observed with an ISE system for measuring TPP, a pH electrode is the usual choice because it is available in many labs. The only requirements for the second electrode is that it can be connected via a BNC port. It does not have to be an electrode from Oroboros Instruments neither is it necessary that the electrode fits into the O2k chamber. However, note that using a pH electrode outside the O2k-Chamber also constitutes a test similar to the one described above (Testing a TPP system outside the O2k-Chamber). Therefore a problem caused e.g. by a leaky POS membrane will not be observed when testing with a pH electrode outside the chamber.<br />
<br />
::: '''Procedure'''<br />
::::* Connect the pH electrode to the oxygraph.<br />
::::* Put the electrode into a pH 4 calibration buffer (event)<br />
::::* Observe (record) the signal in DatLab.<br />
::::* Put the electrode into a pH 7 calibration buffer (event). Obviously the sequence of buffers does not matter.<br />
::::* From such a DatLab file we will see whether the pX electronics is working and even get a very rough estimation of the gain.<br />
<br />
=== Testing TPP or pH electrodes with a voltmeter ===<br />
:::: This test measures the performance of the electrodes independently form the pX electronics of the O2k.<br />
<br />
::: '''Requirements'''<br />
<br />
:::: Any voltmeter, frequently labeled "Multimeter" if different measurement modes can be selected, suitable for measuring mV potentials. .<br />
<br />
::: '''Procedure for TPP electrodes'''<br />
::::* Set up the TPP /reference electrode in the O2k chamber as usually, only medium in the chamber.<br />
::::* Connect the electrodes to the pX electronics and record the signal.<br />
::::* Disconnect both electrode cables from the O2k (but leave the electrodes in the chamber, stirring still on).<br />
::::* If you have a "Multimeter" available set in into "Voltmeter mode" <br />
::::* Set the recording range of the voltmeter to a range good for recording about 200 mV.<br />
::::* Measure the voltage difference between reference electrode and TPP electrode by touching (ideally fixing) the two probes of the voltmeter to the ends of the cables from the electrodes: for the reference electrodes this is simply the gold colored pin, for the TPP electrode this is the INNER , central pin. Note down the voltage reading on the voltmeter display. For this step the help of a second person is most convenient, otherwise you probable have to find a way to fix the cables in some way. It is not necessary to have the voltmeter in contact with the electrodes all the time, just when you are reading the values.<br />
::::* Increase the TPP concentration in a few (lets say 5) rather large steps (at least 2 µM per step) and note down the displayed values.<br />
::::* Finally reconnect the electrodes to the pX electronics of the O2k and observe how much (if at all) the signal has changed there. <br />
::::* Please send us the recorded values along with the TPP concentrations used and the the DatLab file recorded at the same time (that shows the readings for the first and last point). If no change in signal is visible with the voltmeter, the problem is at the TPP electrode, not the O2k.<br />
<br />
::: '''Procedure for pH electrodes'''<br />
<br />
::::* Follow the procedure for TPP electrodes, moving the pH electrode between different pH calibration buffers instead of doing a TPP titration.<br />
<br />
== Ca measurement ==<br />
:::: [[ISE-Ca2+ Membranes]] are not included in the O2k-TPP+ ISE-Module but can be ordered from Oroboros Instruments separately (Product ID 42280-01), for application of the [[O2k-TPP+ ISE-Module| O2k-TPP<sup>+</sup> ISE-Module]] for Ca2+ measurements. <br />
<br />
:::: The Oroboros ion selective electrode (ISE) is designed with replaceable membranes making it possible to measure different ions such as Ca2+, TPP+, TPMP+ with the same electrode housing. See also [[Calcium]] for general consideration about Ca2+ measurements. We recommend to use fluorescence methods for measuring Ca<sup>2+</sup> concentrations, utilizing the [[O2k-Fluo_LED2-Module]]. However, there may be special applications in which determination of Ca<sup>2+</sup> levels via ISE is advantageous. The use of a Ca2+ electrode in mitochondrial research was described by Moreno et al.<ref>Moreno AJM, Vicente JA (2012) Use of a calcium-sensitive electrode for studies on mitochondrial calcium transport. Methods Mol Biol 810:207-17.</ref> Laboratories who are using the [[O2k-TPP+ ISE-Module| O2k-TPP<sup>+</sup> ISE-Module]] (but who do not have the [[O2k-Fluo_LED2-Module]]) may apply the ISE for Ca<sup>2+</sup> detection. <br />
<br />
=== Inner filling solution ===<br />
:::: The following inner filling solution is used: <br />
::::* CaCl<sub>2</sub>: 10 mM<br />
::::* EDTA: 50 mM<br />
::::* pH adjusted to 8.5 with KOH<br />
<br />
=== Conditioning ===<br />
:::: Conditioning of the membrane is a controversial topic. If any conditioning is done, the used free Ca<sup>2+</sup> concentration should probable not be much higher than the highest expected concentration during applications of the electrode.<br />
<br />
=== Calibration ===<br />
:::: As a potentiometric method, the Ca<sup>2+</sup> electrode delivers a signal that is (in the working range) linear to the logarithm of the free Ca<sup>2+</sup> concentration. Therefore, the electrode is calibrated by plotting electrode signal vs. logarithm of the free Ca<sup>2+</sup> concentration. Calibration of the Ca<sup>2+</sup> electrode at low (< 1µM) Ca<sup>2+</sup> levels is typically done by exposing the electrode to a series of Ca<sup>2+</sup> calibration buffers. For a discussion of Ca2+ calibration buffers see [[Calcium#Ca2.2B_calibration]].<br />
<br />
:::: Calculation of free Ca<sup>2+</sup> concentrations:» [[Calcium#Calculation_of_free_Ca2.2B_concentrations]]<br />
<br />
=== Ca2+ electrode and FCCP ===<br />
:::: There are strong indications that FCCP and the [[ISE-Ca2+ Membranes]] are incompatible.<br />
<br />
<br />
== Other ions beside TPP+ TPMP+, and Ca2+ ==<br />
:::: There are ion selective membranes available, e.g. from Sigma Aldrich, for a wide variety of species. If cut to a circle with a diameter of 4 mm these membranes can be used in the ISE system. Many ions may also be detected by fluorimetric based methods, see [[O2k-Fluorescence_LED2-Module]].<br />
<br />
<br />
== References ==<br />
<br />
<references/><br />
<br />
:[[MiPNet15.08 TPP electrode]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet15.05_NO-manual&diff=149145MiPNet15.05 NO-manual2018-01-11T08:17:23Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-MultiSensor system with amperometric sensors: NO, H<sub>2</sub>S, H<sub>2</sub>O<sub>2</sub>.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/d/d3/MiPNet15.05_NO-Manual.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet15.05_NO-Manual.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-03-06<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M, Gnaiger E (2014) O2k-MultiSensor system with amperometric sensors: NO, H<sub>2</sub>S, H<sub>2</sub>O<sub>2</sub>. Mitochondr Physiol Network 15.05(04):1-8.''' - [http://www.bioblast.at/index.php/File:MiPNet15.05_NO-Manual.pdf Versions]<br />
<br />
<br />
'''O2k-Manual:''' Two additional amperometric amplifiers are integrated into the OROBOROS O2k-Core. Two NO sensors can be directly connected, and both chambers can be used simultaneously for oxygen and NO, and two additional potentiometric electrodes can be monitored simultaneously. NO, H<sub>2</sub>O<sub>2</sub> and similar sensors are amperometric sensors, as is the Oroboros polarographic oxygen sensor ([[OroboPOS]]). The measured current is converted to a voltage, amplified and recorded by DatLab.<br />
<br />
:» Product: [[O2k-NO Amp-Module]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=Oxygraph-2k, NO, O2k-Manual<br />
|additional=O2k-MultiSensor, O2k-SOP, DL6, DL7, DL6a7<br />
}}<br />
<br />
{{Template:Technical support integrated}}<br />
<br />
<br />
__TOC__<br />
<br />
=== O2k signal and output ===<br />
:::# [[O2k signals and output#Signal of the O2k-Core and add-on modules |O2k signal]]: The [[O2k-NO Amp-Module]] is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.<br />
:::# [[O2k signals and output#O2k output |O2k output]]: type A and B<br />
<br />
<br />
=== Electrochemical NO and H<sub>2</sub>O<sub>2</sub> sensors ===<br />
:::: NO and H<sub>2</sub>O<sub>2</sub> sensors use an amperometric measurement principle (Amp-Module), comparable to the polarographic oxygen sensor (OroboPOS), which yields an amperometric signal at a given polarization voltage. We supply custom-made stoppers that can accommodate such sensors. All amperometric sensors (NO, H<sub>2</sub>S, H<sub>2</sub>O<sub>2</sub>) that have the same connection can be used with the O2k. <br />
<br />
=== Requirements for NO or other amperometric sensors ===<br />
<br />
<br />
[[File:MiPNet15.05 Layout 01.jpg|250px|left|Figure 1. MultiSensor Features of DatLab 4: NO-Signal - Graph layout]]<br />
<br />
<br />
::::1.NO sensor (not yet available from Oroboros Instruments; see Aguirre et al 2010).<br />
::::2.PVDF stopper to accommodate the NO sensor (#30221-24 for 2 mm shaft NO sensor). <br />
<br />
<br />
::::'''Oxygraph-2k Series A-C'''<br />
::::A.3. NO amplification box (#30430-24).<br />
<br />
<br />
::::'''Oxygraph-2k Series D (and higher)'''<br />
::::D.3. MultiSensor electronic upgrading (#30100-34).<br />
<br />
<br />
<br />
[[File:Questions.jpg|left|50px]]<br />
=== NO-sensor calibration ===<br />
::::* I have a question regarding NO-sensor calibration. What method would you recommend? I just tried with a solution (outside of the Oroboros O2k) recommended from the factory (0.05 M H<sub>2</sub>SO<sub>4</sub>, 0.02 M KI with addition of nitrite in different concentrations). This method worked fine, however, is this possible to do in the O2k? Is that extremely acidic solution harmful to the POS membranes? Best regards ~ [[Schiffer TA]]<br />
# Answer: That is the method we always used. It is fine for the O2k and no problem for the PTFE O2 membranes. But I have to admit that the laboratory table got some "scars" :-) from the KI, and the blue POM chamber holders may change colour. ~ support@oroboros.at, 10 March 2011<br />
<br />
<br />
[[File:Questions.jpg|left|50px]]<br />
=== Pre-polarisation times ===<br />
::::* For some sensors WPI states the suggested pre-polarisation time not in the manual of their sensor but only in the manual of the WPI electronics.<br />
::::* '''User feedback''' for the WPI HPO-2 sensor: ''The suggested polarization voltage for the WPI H<sub>2</sub>O<sub>2</sub> electrodes is 400 mV, that means -400 mV have to be set in the O2k (see [[MiPNet15.05 NO-Manual]]). I have also contacted with WPI. They said that "The ISO-HPO-2 polarized at 450 mV for 2 hours in 0.1 M PBS solution." But I find 2 hours is not enough. I send this information to you, maybe it is helpful for other customers.<br />
::::# '''Comment by Oroboros''': Based on our experience with the WPI NO electrode and our users experience with the WPI HPO electrode, the required prepolarisation time is longer than the (minimum) cited by WPI. Especially for the first run we suggest to allow for a very long pre-polarisation time (minimum over-night, days for NO) and to observe the slope of the signal. Only when the slope of the signal (not the signal itself !) is constant for a long time (hours), pre-polarisation is finished. This will give an indication for the proper pre-polarisation time in further experiments. It is important to continue the pre-polarisation for a few hours after the slope appears to be constant. In the later phase of pre-polarisation the slope may change very slowly but may still approach closer to zero. This can only be detected by recording the slope for a sufficiently long period of time. ~ support@oroboros.at, 23 January 2014<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet15.08_TPP_electrode&diff=149142MiPNet15.08 TPP electrode2018-01-11T08:16:17Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] Ion selective electrode for TPP<sup>+</sup> and high-resolution respirometry. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/c/c5/MiPNet15.08_TPP-electrode.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet15.08_TPP-electrode.pdf Versions]<br />
|authors=Oroboros<br />
|year=2011-12-11<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Sumbalova Z, Fasching M, Gnaiger E (2011) Ion selective electrode for TPP<sup>+</sup> and high-resolution respirometry. Mitochondr Physiol Network 15.8.''' <br />
<br />
Tetraphenylphosphonium (TPP<sup>+</sup>) accumulates in the mitochondrial matrix as a function of the [[mitochondrial membrane potential]]. The TPP<sup>+</sup> electrode is an ion selective electrode (ISE). The voltage signal [V] is linearly dependent on the logarithm of the free concentration [TPP<sup>+</sup>].<br />
:» [http://www.bioblast.at/images/0/05/MiP2010_Sumbalova_Poster.pdf Poster]<br />
:» [[O2k-Protocols]]<br />
:» Product: [[Oroboros O2k]], [[ISE]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|keywords=O2k-MultiSensor System<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|instruments=Oxygraph-2k, TPP, O2k-Protocol<br />
|additional=O2k-Demo, O2k-MultiSensor<br />
}}<br />
:* [[MiPNet15.03 O2k-MultiSensor-ISE]]<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet15.09._Yeast:_HRR_Reference_Assay&diff=149140MiPNet15.09. Yeast: HRR Reference Assay2018-01-11T08:15:45Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] A mitochondrial reference assay for high-resolution respirometry using freeze-dried Baker’s yeast.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/62/MiPNet15.09_mtRA-Yeast.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet15.09_mtRA-Yeast.pdf Versions]<br />
|authors=Oroboros<br />
|year=2017-02-05<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Simonovik B, Gnaiger E (2017) A mitochondrial reference assay for high-resolution respirometry using freeze-dried Baker’s yeast. Mitochondr Physiol Network 15.09(03).'''<br />
<br />
A mitochondrial reference assay (mtRA) was designed for high-resolution respirometry, to evaluate the reproducibility of cell respiration with baker’s yeast (Saccharomyces cerevisiae). Quality control is an integral part of quality assurance (QA), which is becoming increasingly important in high-resolution respirometry for basic and clinical research, as well as industrial applications. Quality control will ensure comparability of quantitative results obtained by different research groups.<br />
:» Product: [[Oxygraph-2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]]<br />
|keywords=Yeast: HRR Reference Assay<br />
|mipnetlab=AT Innsbruck Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Saccharomyces cerevisiae, Fungi<br />
|couplingstates=ROUTINE, ET<br />
|pathways=ROX<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-Demo, O2k-Core<br />
}}<br />
<br />
<br />
[[Category:OroboPedia]]<br />
== See also ==<br />
* O2k-Fluorometry: Amplex® UltraRed using freeze-dried baker’s yeast. - [[MiPNet18.06 Amplex-Yeast |»Bioblast link«]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet17.02_PBI-Shredder_manual&diff=149139MiPNet17.02 PBI-Shredder manual2018-01-11T08:14:41Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=http://wiki.oroboros.at/index.php/O2k-Manual|O2k-Manual]] PBI-Shredder HRR-Set: preparation of tissue homogenates for diagnosis of mitochondrial respiratory function. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/c/c7/MiPNet17.02_PBI-Shredder_HRR-Set.pdf |Bioblast pdf]] » [http://www.bioblast.at/index.php/File:MiPNet17.02_PBI-Shredder_HRR-Set.pdf Versions]<br />
|authors=Oroboros<br />
|year=2013-11-22<br />
|journal=Mitochondr Physiol Network<br />
|abstract=[[Image:PBI-Shredder HRR-Set.JPG|180px|right|link=http://www.bioblast.at/index.php/PBI-Shredder_HRR-Set]]<br />
'''Draxl A, Eigentler A, Gnaiger E (2013) PBI-Shredder HRR-Set: preparation of tissue homogenates for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 17.02(03):1-9.'''<br />
<br />
The PBI-Shredder HRR-Set is an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues with high reproducibility of mitochondrial yield and mitochondrial function. In this manual for applications with high-resolution respirometry (HRR), we refer to the PBI User Manual with safety information, product use limitations and warranty information, and the Product Specification Sheet by Pressure BioSciences Inc. (PBI).<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]<br />
|mipnetlab=AT_Innsbruck_Oroboros, AT_Innsbruck_Gnaiger E, AT Innsbruck MitoCom<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|preparations=Homogenate<br />
|instruments=O2k-Manual, O2k-Protocol<br />
|additional=Auxiliary, Mt-Preparation, PBI-Shredder<br />
}}<br />
__TOC__<br />
== Further information ==<br />
::::» [[PBI-Shredder HRR-Set]]<br />
<br />
== List of publications: PBI-Shredder ==<br />
{{#ask:[[Category:Publications]] [[Additional label::PBI-Shredder]]<br />
|?Was published in year=Year<br />
|?Has title=Reference<br />
|?Mammal and model<br />
|?Tissue and cell<br />
|?Stress <br />
|?Diseases <br />
|format=broadtable<br />
|limit=5000<br />
|offset=0<br />
|sort=Was published in year<br />
|order=descending<br />
}}<br />
<br />
== List of abstracts: PBI-Shredder ==<br />
{{#ask:[[Category:Publications]] [[Additional label::PBI-Shredder]]<br />
|?Was submitted in year=Year<br />
|?Has title=Reference<br />
|?Mammal and model<br />
|?Tissue and cell<br />
|?Stress <br />
|?Diseases <br />
|format=broadtable<br />
|limit=5000<br />
|offset=0<br />
|sort=Was submitted in year<br />
|order=descending<br />
}}<br />
<br />
[[File:PBI-Shredder SG3.jpg|200px|thumb|left|link=]]<br />
[[File:PBI-Shredder HRR-Set.JPG|240px|thumb|left|link=]]<br />
[[File:Shredder-Tube Cap Tool.JPG|240px|thumb|left|link=]]<br />
[[File:Shredder-Tube Analyt Biochem.jpg|300px|thumb|left|link=]]<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet17.03_Shredder_vs_Fibers&diff=149138MiPNet17.03 Shredder vs Fibers2018-01-11T08:14:07Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/8/80/MiPNet17.03_PBI-Shredder_Fish-heart-liver.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet17.03_PBI-Shredder_Fish-heart-liver.pdf Versions]<br />
|authors=Oroboros<br />
|year=2015-06-18<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Doerrier C, Draxl A, Wiethuechter A, Eigentler A, Gnaiger E (2015) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03(04):1-9.''' <br />
<br />
In the present study we compared mitochondrial function of permeabilized fibres and homogenate of heart muscle of mice. In addition, respiration of trout heart homogenate preparations were compared with permeabilized fibres, and the PBI-Shredder was successfully tested with preparation of trout liver.<br />
:» Product: [[Oxygraph-2k |Oroboros O2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]]<br />
|keywords=Tissue homogenate, Shredder<br />
|mipnetlab=AT_Innsbruck_Oroboros, AT Innsbruck MitoCom<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods, Comparative MiP;environmental MiP<br />
|organism=Fishes<br />
|tissues=Heart, Liver<br />
|preparations=Homogenate<br />
|couplingstates=LEAK, OXPHOS, ET<br />
|pathways=N, S, NS<br />
|instruments=Oxygraph-2k, O2k-Protocol<br />
|additional=O2k-Demo, Mt-Preparation, Auxiliary, PBI-Shredder, O2k-Core<br />
}}<br />
== Further information ==<br />
:» [[PBI-Shredder HRR-Set]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet17.04_CitrateSynthase&diff=149137MiPNet17.04 CitrateSynthase2018-01-11T08:13:39Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Laboratory protocol: Citrate synthase. Mitochondrial marker enzyme. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/40/MiPNet17.04_CitrateSynthase.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet17.04_CitrateSynthase.pdf Versions]<br />
|authors=Oroboros<br />
|year=2015-06-18<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Eigentler A, Draxl A, Wiethüchter A, Kuznetsov AV, Lassnig B, Gnaiger E (2015) Laboratory protocol: Citrate synthase. Mitochondrial marker enzyme. Mitochondr Physiol Network 17.04(03):1-11.''' <br />
<br />
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT Innsbruck Oroboros<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods, mt-Biogenesis;mt-density<br />
|preparations=Enzyme<br />
|enzymes=Marker enzyme<br />
|instruments=O2k-Protocol<br />
|additional=Mt- and marker-enzymes<br />
}}<br />
== Further information ==<br />
<br />
:» Continue the discussion:[[Talk:MiPNet17.04 CitrateSynthase]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet17.05_O2k-Fluo_LED2-Module&diff=149136MiPNet17.05 O2k-Fluo LED2-Module2018-01-11T08:13:11Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-Fluo LED2-Module.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/9e/MiPNet17.05_O2k-Fluo_LED2-Module.pdf |Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:MiPNet17.05_O2k-Fluo_LED2-Module.pdf Versions]<br />
|authors=Oroboros<br />
|year=2016-08-09<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M, Gradl P, Gnaiger E (2016) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(09):1-6.''' <br />
<br />
<br />
'''O2k-Manual:''' The O2k-Fluo LED2-Module is a modular extension of the O2k-Core (O2k-Series D to G). A growing number of fluorescence markers enables determination of diverse mitochondrial processes in addition to oxygen consumption, including generation of H<sub>2</sub>O<sub>2</sub>, ATP production, mitochondrial membrane potential and Ca<sup>2+</sup>, extendable by user-specific applications.<br />
:» Product: [[O2k-Fluo LED2-Module]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]<br />
|keywords=[[HRR]], [[Fluorometry]]<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Manual, O2k-Protocol<br />
|additional=O2k-MultiSensor<br />
}}<br />
<br />
<br />
<br />
<br />
<br />
<br />
* Contribution to K-Regio project ''MitoCom Tyrol'', funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).&nbsp;&nbsp;&nbsp; >> [[MitoCom_O2k-Fluorometer|''MitoCom O2k-Fluorometer'']]<br />
<br />
{{Template:Technical support integrated}}<br />
<br />
<br />
__TOC__<br />
<br />
== O2k-Manual: O2k-Fluo LED2-Module ==<br />
<br />
=== Setup of the O2k-Fluo LED2-Module ===<br />
:::# [[Fluorescence-Control_Unit#Setup_of_the_O2k-Fluorescence_LED2-Module|Setup of the O2k-Fluo LED2-Module]]<br />
:::# [[Fluorescence-Sensor#Select_the_Fluorescence-Sensors|Selecting a Fluorescence Sensor]]'''<br />
:::# [[Filter-Cap#Mounting_a_Filter-Cap|Mounting a Filter-Cap]]<br />
:::# [[Fluorescence-Sensor#Connect_Fluorescence-Sensor_to_O2k|Connect Fluorescence-Sensor to O2k-Main unit]]<br />
:::# [[Fluorescence-Control_Unit#Power_on|Power on]]<br />
<br />
<br />
=== LED-intensity and amplification ===<br />
:::# [[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]<br />
:::# [[Fluorescence-Control_Unit#Gain|Amplification]]<br />
<br />
::::The light intensity of the LED ([[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]) and the signal amplification ([[Fluorescence-Control_Unit#Gain|Gain]]) can be adjusted in a wide range. The table suggests initial values, which can be optimised for specific applications. <br />
::::* The settings depend on the concentration of the fluorophore, which vary between different applications. Therefore, only recommendations for specific fluorophore concentrations are given. In the Amplex UltraRed assay the fluorophore is formed during the experiment.<br />
::::* The recommendations apply to experiments at 37 °C. The Fluorescence intensity increases strongly at lower temperatures. Then the light intensity is reduced to avoid off-scale signals. <br />
<br />
<br />
== Fluorophores ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Application !!Sensor!! Filter set !! O2k output !! Light intensity (polarization voltage) - ''Note a'' !! Gain !! Comment<br />
|- <br />
| [[Amplex UltraRed]]||[[Fluorescence-Sensor Green]] || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type B]] || 100 - 500 || 1000 (at light intensity = 100) || <br />
|- <br />
| [[TMRM]]||[[Fluorescence-Sensor Green]] || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type C]] || 200 - 500|| 1000 || at c(TMRM) = 2 µM<br />
|-<br />
| [[Safranin]] ||[[Fluorescence-Sensor Blue]]|| [[Filter Set Saf|Saf]] || [[O2k_signal_and_output| Type C]] || 100 - 200 for c(safranin)= 2 µM; || 1000 || at c(Mg Green) = 2 µM, <br />
|-<br />
| [[Magnesium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output| Type B]] || 100 - 300|| 1000 || at c(Mg Green) = 2 µM<br />
|-<br />
| [[Calcium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output|Type A and C]] ||100 -300|| 1000 ||at c(Ca Green) = 2 µM<br />
|}<br />
<br />
::::* ''Note a'': Set the polarization voltage [mV] for the amperometric channel (Amp) in the DatLab menu [O2k-MultiSensor \ O2k Control \ Amp polarisation voltage]. Divide the polarisation voltage [mV] by 100 to obtain the current [mA] through the LED. For simple operation instructions, it is sufficient to refer to the polarization settings selected in DatLab.<br />
<br />
<br />
=== TMRM with the [[O2k-Fluo LED2-Module]]: Experiments with permeabilized HEK 293T cells by M. Hansl and G. Krumschnabel ===<br />
<br />
::: '''HEK 293T cells'''<br />
<br />
:::: 1.5 10<sup>6</sup> cells /ml <br />
<br />
:::: Experimental buffer: MiR05<br />
<br />
:::: Pyruvate: 5 mM; Malate: 2 mM; ADP: 2.5 mM; Succinate: 10 mM; CCCP (U) added in 0.5-1 µM steps; Rotenone: 1 µg/ml; Antimycin A: 2.5µM; TMRM: 2 µM final concentration<br />
<br />
:::: '''Experimental conditions:'''<br />
:::: 2 ml chamber volume, [[Fluorescence-Sensor_Green|Fluorescence-Sensor Green]], [[Filter_Set_AmR|Filter Set AmR]], light intensity: Amp Polarization voltage = 500 , gain 1000, T = 37.0 °C <br />
<br />
<br />
[[Image:TMRM_permHEK293T.png|600px]]<br />
<br />
:::: '''Figure legend:''' Simultaneous measurement of respiration [upper panel; pmol/(s*ml)] and mitochondrial membrane potential (mtMP) (lower panel; arbitrary units) in permeabilized HEK 293T cells: TMRM was added to the O2k chamber with 2 ml experimental buffer in two 1 µl steps to obtain 2 µM TMRM and then 1.5 10<sup>6</sup> cells/ml were added. Next, cells were permeabilized by adding 10 µg/ml digitonin and this was followed by addition of pyruvate and malate to induce LEAK respiration. Then, 2.5 mM ADP was added to elicit CI-linked OXPHOS, then succinate was injected to obtain CI&II-linked OXPHOS, before titrating uncoupler CCCP (U) in 0.5-1 µM steps to induce fully uncoupled respiration and fully collapsed mtMP. It should be noted that in the permeabilized cells CCCP concentration required for complete collapse of mtMP was lower than that needed for maximum respiration. Finally, CI-inhibitor rotenone (Rot) and CIII-inhibitor antimycin A (Ama) were added to obtain residual oxygen consumption (ROX), which however did not further affect mtMP (further details: [[Talk:TMRM]]).<br />
<br />
<br />
=== The fluorescence signal ===<br />
:::# [[O2k signals and output#Signal of the O2k-Core and add-on modules |O2k signal]]: The [[O2k-Fluo LED2-Module]] is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [Amp]) as the primary signal.<br />
:::# [[O2k signals and output#O2k output |O2k output]]: type A, B, or C, or combinations.<br />
<br />
[[Image:DL7graph_select_plots_amp.jpg|thumb|250px|alt=Check Amp raw signal form Graph/Select Plots to display the fluorometric signal| Graph / Select plots ]]'''Graph layout''': Three plots are available in DatLab based on the recorded signal: '''Amp Raw Signal''', '''Amp Calibrated''', and '''Amp Slope'''<nowiki>. These plots can be selected from the drop-down lines and displayed with their check boxes either on the Y1 or Y2 [Graph layout / Select Plots].</nowiki> <br />
<br />
<br />
:::: '''Amp Raw Signal''' displays the raw voltage (including amplification) as recorded by the O2k at a given gain setting. <br />
<br />
:::: '''Amp Calibrated''' is the signal after calibration with the parameters set in the O2k-MultiSensor Calibration window. <br />
<br />
:::: '''Amp slope''' is the time derivative of the '''calibrated''' signal, multiplied by '''1000''', in units [mV(conc. Unit during calibration)/s], so if the signal was calibrated in µM [nmol/ml] the unit of the slope is pmol/(s ml). To obtain the slope of the raw signal check the appropriate box in the calibration window ([[DatLab]] 5.1.0.130 and above).<br />
<br />
:::: '''Graphs''' can be generated to display oxygen and fluorescence data, or several graphs can be added to display oxygen and fluorescence data separately. Layout templates are provided, which can be modified and saved as appropriate. All graph settings can be saved as user-defined layouts, see [[MiPNet19.01C DatLab Guide]].<br />
<br />
<br />
<br />
== O2k-Fluorometry and the TIP2k ==<br />
::::* [[Titration-Injection microPump |TiP2k]]: Our tests indicated that the fluorometric signal is not affected by the TIP2k needle inserted into the O2k-Chamber.</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet17.15_PBI-Shredder_Mouse-heart-brain-liver&diff=149135MiPNet17.15 PBI-Shredder Mouse-heart-brain-liver2018-01-11T08:07:53Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] Tissue homogenates for diagnosis of mitochondrial respiratory function: Mouse heart, brain and liver.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/c/c9/MiPNet17.15_PBI-Shredder_Mouse-heart-brain-liver.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet17.15_PBI-Shredder_Mouse-heart-brain-liver.pdf Versions]<br />
|authors=Oroboros<br />
|year=2015-06-16<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Eigentler A, Fontana-Ayoub M, Gnaiger E (2015) Tissue homogenates for diagnosis of mitochondrial respiratory function: Mouse heart, brain and liver. Mitochondr Physiol Network 17.15(04):1-6.''' <br />
<br />
A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (e.g. heart, liver, brain) with high reproducibility of 100% mitochondrial yield and mitochondrial function as evaluated with HRR.<br />
:>> O2k-Protocols:[[O2k-Protocols| Overall contents]]<br />
:>> Product: [[Oroboros O2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]]<br />
|keywords=Tissue homogenate, Shredder<br />
|mipnetlab=AT_Innsbruck_Oroboros, AT Innsbruck MitoCom<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Mouse<br />
|tissues=Heart, Nervous system, Liver<br />
|preparations=Homogenate<br />
|couplingstates=LEAK, OXPHOS, ET<br />
|pathways=N, S, NS<br />
|instruments=Oxygraph-2k, O2k-Manual, O2k-Protocol<br />
|additional=Mt-Preparation, PBI-Shredder<br />
}}<br />
== Further information==<br />
:>> [[PBI-Shredder HRR-Set]]<br />
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[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet17.16_O2k-Spectrophotometry&diff=149134MiPNet17.16 O2k-Spectrophotometry2018-01-11T08:07:18Z<p>Hiller Elisabeth: </p>
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<div>{{OROBOROS header page name}}<br />
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{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k-Spectrophotometry - A MitoCom Project. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/9/95/MiPNet17.16_O2k-Spectrophotometry.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet17.16_O2k-Spectrophotometry.pdf Versions]<br />
|authors=Oroboros<br />
|year=2015-06-16<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Harrison DK, Fasching M, Gnaiger E (2015) O2k-Spectrophotometry - A MitoCom Project. Mitochondr Physiol Network 17.16(02):1-4.''' <br />
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The principal thrust of the technical development of the O2k high-resolution respirometer within the MitoCom project is towards the O2k-Fluorometer. However, simultaneously, a parallel project is working towards the integration of spectrophotometry into the O2k in order to measure the redox state of cytochromes – particularly cytochromes ''c'' and ''aa''3, but also cytochrome ''b''.<br />
|keywords=O2k, High-resolution respirometry, Spectrophotometry, MitoCom<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|instruments=Oxygraph-2k, O2k-Spectrophotometer, O2k-Protocol<br />
|additional=O2k-Demo, O2k-MultiSensor<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet17.17_Amplex-Mouse-brain&diff=149133MiPNet17.17 Amplex-Mouse-brain2018-01-11T08:06:43Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k-Fluorometry: HRR and H<sub>2</sub>O<sub>2</sub> production in mouse brain mitochondria.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/e/e2/MiPNet17.17_Amplex-Mouse-brain.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet17.17_Amplex-Mouse-brain.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-11-14<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H<sub>2</sub>O<sub>2</sub> production in mouse brain mitochondria. Mitochondr Physiol Network 17.17.''' <br />
:» Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. [[Krumschnabel 2015 Methods Mol Biol |»Bioblast pdf«]]<br />
|mipnetlab=AT_Innsbruck_Oroboros, AT Innsbruck MitoCom<br />
}}<br />
{{Labeling<br />
|additional=Archive<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet18.05_Amplex-Mouse-heart&diff=149132MiPNet18.05 Amplex-Mouse-heart2018-01-11T08:05:46Z<p>Hiller Elisabeth: </p>
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<div>{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k-Fluorometry: HRFR and H<sub>2</sub>O<sub>2</Sub> production in mouse cardiac tissue homogenate.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/2a/MiPNet18.05_Amplex-Mouse-heart.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet18.05_Amplex-Mouse-heart.pdf Versions]<br />
|authors=Oroboros<br />
|year=2015-06-16<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Eigentler A, Fontana-Ayoub M, Gnaiger E (2015) O2k-Fluorometry: HRFR and H<sub>2</sub>O<sub>2</Sub> production in mouse cardiac tissue homogenate. Mitochondr Physiol Network 18.05(02):1-6.''' <br />
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The [[O2k-Fluorescence LED2-Module]] is an extension of the Oroboros O2k for combining High-Resolution FluoRespirometry and fluorometric measurements. We applied the Fluo-Sensor Green for measurement of H<sub>2</sub>O<sub>2</Sub> production with Amplex® Ultrared in mouse heart mitochondria. Experiments are selected to demonstrate the importance of applying ET-pathway competent substrate states, which are critically evaluated by respiratory performance and considering experimental oxygen levels when measuring mitochondrial H<sub>2</sub>O<sub>2</Sub> production.<br />
:>> Product: [[Oroboros O2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]]<br />
:>> [[Permeabilized muscle fibres]]<br />
|mipnetlab=AT Innsbruck Oroboros, AT Innsbruck MitoCom<br />
}}<br />
{{Labeling<br />
|area=Respiration<br />
|organism=Mouse<br />
|tissues=Heart<br />
|preparations=Homogenate<br />
|topics=Cyt c<br />
|couplingstates=LEAK, OXPHOS, ET<br />
|pathways=N, S, NS<br />
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Protocol<br />
|additional=O2k-Demo, O2k-MultiSensor, Mt-Preparation, PBI-Shredder<br />
}}</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet18.06_Amplex-Yeast&diff=149131MiPNet18.06 Amplex-Yeast2018-01-11T08:05:08Z<p>Hiller Elisabeth: </p>
<hr />
<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k-Fluorometry: Amplex® UltraRed using freeze-dried baker’s yeast.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/f/f9/MiPNet18.06_Amplex-Yeast.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet18.06_Amplex-Yeast.pdf Versions]<br />
|authors=Oroboros<br />
|year=2015-06-15<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Fontana-Ayoub M, Fasching M, Eigentler A, Laner V, Gnaiger E (2015) O2k-Fluorometry: Amplex® UltraRed using freeze-dried baker’s yeast. Mitochondr Physiol Network 18.06(04):1-4''' <br />
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Respiration and hydrogen peroxide production of yeast was studied under hypoxic and hyperoxic conditions, and after stimulation of respiration by glucose using the [[O2k-Fluorescence LED2-Module]]. Extracellular H<sub>2</sub>O<sub>2</sub>-production by intact yeast cells was a pronounced function of environmental oxygen concentration (Fig. 1). Oxygen kinetics of respiration resembled closely but not fully that of isolated mitochondria, indicating that extracellular oxygen levels provide a good approximation of intracellular conditions, and intracellular oxygen gradients are small. Stimulation of respiration was pronounced with the extracellular substrates glucose and ethanol, and with uncoupler titrations. In contrast, these additions exerted a minor effect on extracellular H<sub>2</sub>O<sub>2</sub> production (Fig. 2).<br />
:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
|mipnetlab=AT Innsbruck Oroboros, AT Innsbruck MitoCom<br />
}}<br />
{{Labeling<br />
|area=Respiration<br />
|organism=Saccharomyces cerevisiae, Fungi<br />
|preparations=Intact cells<br />
|topics=Oxygen kinetics, Uncoupler<br />
|couplingstates=ROUTINE, ET<br />
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Protocol<br />
|additional=O2k-Demo, O2k-MultiSensor<br />
}}<br />
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== See also ==<br />
* A mitochondrial reference assay for high-resolution respirometry using freeze-dried Baker’s yeast. - [[MiPNet15.09. Yeast: HRR Reference Assay |»Bioblast link«]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet18.10_O2k-Specifications&diff=149130MiPNet18.10 O2k-Specifications2018-01-11T08:04:31Z<p>Hiller Elisabeth: </p>
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<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:Logo OROBOROS INSTRUMENTS.jpg|right|60px|link=http://www.oroboros.at|OROBOROS]] O2k-FluoRespirometer: specifications for respirometry and comprehensive OXPHOS analysis.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/d/db/MiPNet18.10_O2k_specifications.pdf|Bioblast pdf]] » [http://www.bioblast.at/index.php/File:MiPNet18.10_O2k_specifications.pdf Versions]<br />
|authors=Oroboros<br />
|year=2017-08-08<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Gnaiger E (2017) O2k-FluoRespirometer: specifications for respirometry and comprehensive OXPHOS analysis. Mitochondr Physiol Network 18.10(07):1-8.''' <br />
|keywords=O2k-Specifications<br />
|mipnetlab=AT Innsbruck Oroboros<br />
}}<br />
{{Labeling<br />
|area=Respiration, Instruments;methods<br />
|organism=Human<br />
|tissues=Fibroblast<br />
|preparations=Intact cells, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP<br />
|instruments=Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, Theory<br />
}}<br />
<br />
[[Category:OroboPedia]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet19.01_O2k-Core_Manual_Series_F_DatLab_5&diff=149129MiPNet19.01 O2k-Core Manual Series F DatLab 52018-01-11T08:03:52Z<p>Hiller Elisabeth: </p>
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<div>{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-Core Manual - up to O2k-Series F and DatLab 5. <br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/7/7b/MiPNet19.01_O2k-Core_Manual.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet19.01_O2k-Core_Manual.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-03<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Oroboros (2014) O2k-Core Manual. Mitochondr Physiol Network 19.01(01):1-60.''' <br />
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While the Oroboros O2k provides the instrumental basis for high-resolution respirometry, successful operation at high accuracy up to the limit of detection depends on a professional application by the technician, scientist or student. The O2k-Manual provides professional help and keeps you alert of new developments and updated DatLab features.<br />
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>> Product: [[Oxygraph-2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]<br />
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|keywords=HRR<br />
}}<br />
{{Labeling<br />
|area=Instruments;methods<br />
|instruments=Oxygraph-2k, O2k-Manual<br />
|additional=DatLab, Archive<br />
}}<br />
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:::<big>'''Contents: O2k-Core Manual up to Series F and DatLab 5'''</big><br />
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:::: A: [[MiPNet19.01A O2k-Start |Oxygraph-2k: start high-resolution respirometry.]]<br />
:::: B: [[MiPNet19.01B POS-Service |Service of the polarographic oxygen sensor OroboPOS.]]<br />
:::: C: [[MiPNet19.01C DatLab Guide |DatLab guide through the menus.]]<br />
:::: D: [[MiPNet19.01D O2k-Calibration |O2k-calibration by DatLab.]]<br />
:::: E: [[MiPNet19.01E O2 Flux Analysis |Oxygen flux analysis: DatLab real-time.]]<br />
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:::<big>'''Contents: O2k-Core Manual Series G and DatLab 6'''</big><br />
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::::» [[MiPNet19.18 O2k-Core Manual]]</div>Hiller Elisabethhttps://wiki.oroboros.at/index.php?title=MiPNet19.01A_O2k-Start&diff=149128MiPNet19.01A O2k-Start2018-01-11T08:03:14Z<p>Hiller Elisabeth: </p>
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<div>{{OROBOROS header page name}}<br />
{{Publication<br />
|title=[[Image:O2k-Manual.jpg|right|70px|link=http://wiki.oroboros.at/index.php/O2k-Manual|O2k-Manual]] O2k: start high-resolution respirometry.<br />
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/28/MiPNet19.01A_O2k-Start.pdf|Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet19.01A_O2k-Start.pdf Versions]<br />
|authors=Oroboros<br />
|year=2014-03<br />
|journal=Mitochondr Physiol Network<br />
|abstract='''Gnaiger E, Fasching M, Gradl L, Gradl P (2014) O2k: start high-resolution respirometry. Mitochondr Physiol Network 19.01(A01):5-18.''' <br />
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'''This is an old version, which applies up to O2k-Series F and to DatLab 5.'''<br />
: ''New version:'' '''[[MiPNet19.18A O2k-start|»MiPNet19.18A O2k-start]]'''<br />
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:>> Product: [[Oxygraph-2k|O2k]], [[Oroboros O2k-Catalogue]]<br />
|keywords=HRR<br />
|mipnetlab=AT_Innsbruck_Oroboros<br />
}}<br />
{{Labeling<br />
|additional=Archive<br />
}}<br />
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* '''O2k-Core Manual Series F, DatLab 5 »[[MiPNet19.01 O2k-Core Manual Series F DatLab 5|Contents]]'''<br />
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* '''New version: »[[MiPNet19.18_O2k-Core_Manual|Contents]]'''<br />
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[[Category:OroboPedia]]</div>Hiller Elisabeth