Difference between revisions of "SUIT-013"

Latest revision as of 09:38, 9 November 2021

high-resolution terminology - matching measurements at high-resolution

SUIT-013

Description

Abbreviation: ce

Reference: A

SUIT-category: ce

SUIT protocol pattern: ce1

SUIT-013 has been designed to study the oxygen dependence of H₂O₂ production in intact cells. SUIT-013 AmR ce D023 specifically has been tested for demonstration of the oxygen dependence in baker´s yeast. For more details see: Komlodi 2021 MitoFit AmR-O2. The respiration medium has to be choosen carefully since the Amplex UltraRed assay leads to artefact formation with the components of the buffer.

Communicated by  Komlodi T and Gnaiger E  (last update 2021-11-09)

Specific DLP protocols

SUIT-013 AmR ce D023

SUIT-013 AmR ce D023 for intact cells

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
0DTPA				DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD				SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H₂O₂, making it accessible to the Amplex UltraRed assay.
0HRP				HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H₂O₂ towards the fluorescent resorufin.
0AmR				AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
ce1	ROUTINE			ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: SUIT 003 AmR ce D058 with AmR and SUIT 003 AmR ce D059 with carrier titration as a control. These two protocols have to be done in parallel.

+ Reasonable duration of the experiment.

+ Quick demonstration of the oxygen dependence of H₂O₂ production in intact cells.

+ No externally added substrates are required with yeast.

Compare SUIT protocols

References

	Year	Reference	Organism	Tissue;cell
MiPNet24.10 H2O2 flux analysis	2021-10-22	Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
Komlodi 2021 BEC AmR-O2	2021	Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004	Saccharomyces cerevisiae	Other cell lines
MiPNet20.14 AmplexRed H2O2-production	2019-06-24	O2k-FluoRespirometry: HRR and simultaneous determination of H₂O₂ production with Amplex UltraRed.	Mouse	Heart
Komlodi 2018 Methods Mol Biol	2018	Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.	Human Mouse	Skeletal muscle HEK
Makrecka-Kuka 2015 Biomolecules	2015	Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319	Human Mouse	Heart Nervous system HEK
Krumschnabel 2015 Methods Mol Biol	2015	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.	Mouse	Nervous system

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Fluorometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=NS(GM)
+|abbr=ce
-|description=
+|description=[[File:SUIT013 AmR ce D023.png|300px]]
-|info='''A''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-013.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:SUIT-013.pdf Versions]
+|info='''A'''
 }}
+::: '''[[Categories of SUIT protocols |SUIT-category]]:''' ce
-::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(GM)
+::: '''[[SUIT protocol pattern]]:''' ce1
-::: '''[[SUIT protocol pattern]]:''' orthogonal 1GMS;2D;3Ama
+SUIT-013 has been designed to study the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in intact cells. SUIT-013 AmR ce D023 specifically has been tested for demonstration of the oxygen dependence in baker´s yeast. For more details see: [[Komlodi 2021 MitoFit AmR-O2]]. The respiration medium has to be choosen carefully since the [[Amplex UltraRed]] assay leads to artefact formation with the components of the buffer.
 __TOC__
-  Communicated by  [[Komlodi T]]  (last update 2019-01-22)
+  Communicated by  [[Komlodi T]] and [[Gnaiger E]]  (last update 2021-11-09)
-== DLP applications ==
+== Specific DLP protocols ==
+=== SUIT-013 AmR ce D023 ===
-== References ==
+[[File:SUIT013 AmR ce D023.png|200px]] [[File:SUIT-013 O2 D023.png|600px]]
-{{#ask:[[Category:Publications]] [[Additional label::SUIT-013]]  [[Additional label::O2k-Protocol]]
+[[File:SUIT-013 AmR D023.png|600px]]
-| ?Was published in year=Year
+:::*[[SUIT-013 AmR ce D023]] for intact cells
-| ?Has title=Reference
-| ?Mammal and model=Organism
-| ?Tissue and cell=Tissue;cell
-| format=broadtable
-| limit=5000
-| offset=0
-| sort=Was published in year
-| order=descending
-}}
 {{Template:SUIT-013}}
 == Strengths and limitations ==
+:::* To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: [[SUIT 003 AmR ce D058]] with AmR and [[SUIT 003 AmR ce D059]] with carrier titration as a control. These two protocols have to be done in parallel.
-:::* A succinate concentration > 10 mM might be required to reach saturating S-OXPHOS capacity.
-:::* In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.
-:::+ Contribution of N pathway to the NS can be checked after the addition of Rot.
 :::+ Reasonable duration of the experiment.
-:::+ This protocol can be extended with the Complex IV module.
+:::+ Quick demonstration of the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in intact cells.
-:::- Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).
+:::+ No externally added substrates are required with yeast.
-:::- Careful washing is required after the experiment to avoid carry-over of inhibitors.
 == Compare SUIT protocols ==
+== References ==
+{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-013]]
+|?Was published in year=Year
+|?Has title=Reference
+|?Mammal and model=Organism
+|?Tissue and cell=Tissue;cell
+|format=broadtable
+|limit=5000
+|offset=0
+|sort=Was published in year
+|order=descending
+}}
 {{MitoPedia concepts
@@ Line 46: / Line 43: @@
 }}
 {{MitoPedia methods
-|mitopedia method=Respirometry, Fluorometry
+|mitopedia method=Fluorometry
 }}
-{{MitoPedia O2k and high-resolution respirometry}}
-{{MitoPedia topics}}