MiPNet14.13 Medium-MiR06: Difference between revisions
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= Further information = | = Further information = | ||
== MiR07 == | * [[MiR07]] = MiR06 + Creatine | ||
== Application of MiR06 in [[HRR]] == | |||
'''MiR06: Mitochondrial Respiration Medium''' ([[#Preparation of MiR05 (MiR06) stock solution|MiR06]] = [[MiR05]] + [[Catalase]]). Total volume = 1 litre. | |||
::Oxygen solubility factor in MiR06 (MiR06) at 30°C and 37°C = 0.92 | |||
::pH of MiR05/06: 7.2 (20°C), 7.2 (25°C), 7.1 (30°C), 7.1 (35°C), 7.0 (37°C) | |||
'''To increase oxygen levels''' small volumes (µl) of [[#Preparation of 200 mM H2O2 stock solution|200 mM H<sub>2</sub>O<sub>2</sub> stock concentration]] are injected into the O2k-chamber filled with MiR06. | |||
The '''oxygen solubility factor''' of MiR06 is the same as for [[MiR05|MiR05]] = '''0.92'''. | |||
== Preparation of MiR05 (MiR06) stock solution == | |||
::1) weigh given amounts of the [[#Chemicals for MiR05|listed chemicals]] (except BSA and lactobionic acid) and transfer to a 1000 ml glass beaker | |||
::2) disrupt big lumps mechanically. It is recommended to do this before adding water, because during dissolution these lumps do not disintegrate easily. | |||
::3) add ~800 ml H<sub>2</sub>O and dissolve on a magnetic stirrer at ~30 °C | |||
::4) add 120 ml of [[#Preparation of K-lactobionate stock solution|K-lactobionate stock solution]] | |||
::5) adjust the pH to 7.1 with 5 N KOH at 30 °C | |||
::6) transfer the MiR05 stock solution to a 1000 ml volumentric glass flask and add H20 to a final volume of 1000 ml <span style="color:#43CD80"> = '''MiR05''' </span> | |||
::7) check pH again and adjust if necessary with small amounts of 5 N KOH | |||
::8) 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) | |||
::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 1 L MiR05 (280 units / ml final concentration) | |||
::10) divide into 40 ml portions and store at -20 °C in plastic vials | |||
::<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 Oxygraph-2k chamber = 280 u/ml. | |||
==== Chemicals for MiR05/MiR06 ==== | |||
<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span> | |||
{|border="1" cellpadding="3" cellspacing="0" | |||
|+ | |||
!Compound | |||
!Final conc. | |||
!FW | |||
!Addition to 1 litre final volume | |||
!Company, product code and storage | |||
|- | |||
|align="right"|EGTA | |||
|align="right"|0.5 mM | |||
|align="right"|380.4 | |||
|align="right"|0.190 g | |||
|align="right"|Sigma E 4378, 25 g, store at R.T. | |||
|- | |||
|align="right"|MgCl<sub>2</sub>.6H<sub>2</sub>0 | |||
|align="right"|3 mM | |||
|align="right"|203.3 | |||
|align="right"|0.610 g | |||
|align="right"|Scharlau MA 0036, 250 g, store at R.T. | |||
|- | |||
|align="right"|Taurine | |||
|align="right"|20 mM | |||
|align="right"|125.1 | |||
|align="right"|2.502 g | |||
|align="right"|Sigma T 0625, 25 g, store at R.T. | |||
|- | |||
|align="right"|KH<sub>2</sub>PO<sub>4</sub> | |||
|align="right"|10 mM | |||
|align="right"|136.1 | |||
|align="right"|1.361 g | |||
|align="right"|Merck 104873, 1000 g, store at R.T. | |||
|- | |||
|align="right"|HEPES | |||
|align="right"|20 mM | |||
|align="right"|238.3 | |||
|align="right"|4.77 g | |||
|align="right"|Sigma H 7523, 250 g, store at R.T. | |||
|- | |||
|align="right"|D-Sucrose | |||
|align="right"|110 mM | |||
|align="right"|342.3 | |||
|align="right"|37.65 g | |||
|align="right"|Roth, 4621.1, 1000 g, store at R.T. | |||
|- | |||
|align="right"|BSA essentially fatty acid free | |||
|align="right"|1 g/l | |||
|align="right"| | |||
|align="right"|1 g | |||
|align="right"|Sigma A 6003 fraction V, 25 g, store at 2-8 °C | |||
|- | |||
|align="right"|Lactobionic acid | |||
|align="right"|60 mM | |||
|align="right"|358.3 | |||
|align="right"|120 ml of 0.5 M [[#Preparation of K-lactobionate stock solution|K-lactobionate stock]] | |||
|align="right"|Aldrich 153516 or Sigma L2398, 100 g, store at R.T. | |||
|- | |||
|} | |||
==== Preparation of K-lactobionate stock solution ==== | |||
::1) weigh 35.83 g lactobionic acid into a 250 ml glass beaker | |||
::2) add 100 ml H<sub>2</sub>O and dissolve by stirring on magnetic stirrer | |||
::2) check pH (is approx. 2.0) and neutralize with 5 N KOH | |||
::4) adjust final volume to 200 ml with H<sub>2</sub>O. It is best to use a 200 ml volumetric glass flask. | |||
::5) check pH again and adjust to 7 if necessary (5 N KOH) | |||
=== Preparation of catalase stock solution === | |||
::'''Catalase''' lypophilized powder, 2000-5000 '''units*'''/mg, Sigma C 9322, store at -20 °C | |||
::'''Stock solution:''' 112000 u/ml (dissolved in MiR05) | |||
::'''Example:''' 'Catalase lypophilized powder, 2800 units/mg solid and 3500 units/mg protein' | |||
::1) Use 'units/mg solid' for your calculations | |||
::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. | |||
::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. | |||
'''Unit definition:''' '''* Units''' of enzymatic activitiy (u) in µmol/min; assay used by Sigma Aldrich: ' ''One unit will decompose 1.0 μmole 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>.'' ' | |||
=== Preparation of 200 mM H2O2 stock solution === | |||
'''H<sub>2</sub>O<sub>2</sub>''': Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge, please see e.g. [http://www.solvayasiapacific.com/static/wma/pdf/1/8/5/8/1/Hydrogen%20Peroxide%20Handling%20%20Storage%201%28English%20version%29.pdf this link] for handling and savety instructions concerning hydrogen peroxide. | |||
::1) pipette 114 µl of 17.6 M H<sub>2</sub>O<sub>2</sub> into 10 ml plastic vial | |||
::2) add H<sub>2</sub>O to a total volume of 10 ml | |||
::3) wrap plastic vial in aluminium foil (solution is light sensitive) and store in the fridge | |||
::4) during experiments keep the solution on ice | |||
'''titration''' of 3 µl of H<sub>2</sub>O<sub>2</sub> into 2 ml chamber would increase the concentration of O<sub>2</sub> by approx. 150 nmol/ml | |||
'''For detailed information on chemicals see [http://www.oroboros.at/index.php?protocols_miro6 MiPNet14.13].''' | |||
=== Preparation of MiR07 === | |||
::1) prepare every morning fresh by adding 3 mg/mL creatine monohydrate (Fluka 27900) to [[MiR06]] | |||
::2) stirr gently on a magnetic stirrer | |||
== Limitations of using MiR05 to MiR07 == | == Limitations of using MiR05 to MiR07 == |
Revision as of 12:08, 8 June 2012
Fasching M, Renner K, Gnaiger E. Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13. |
Fasching M, Renner K, Gnaiger E (
Abstract: Mitochondrial respiration medium 06, developed for oxygraph incubations of mitochondrial preparations = MiR05 plus catalase.
• O2k-Network Lab: AT_Innsbruck_OROBOROS
Labels:
HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property., MiPNet-Publication"MiPNet-Publication" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
Chemicals & Media
Further information
- MiR07 = MiR06 + Creatine
Application of MiR06 in HRR
MiR06: Mitochondrial Respiration Medium (MiR06 = MiR05 + Catalase). Total volume = 1 litre.
- Oxygen solubility factor in MiR06 (MiR06) at 30°C and 37°C = 0.92
- pH of MiR05/06: 7.2 (20°C), 7.2 (25°C), 7.1 (30°C), 7.1 (35°C), 7.0 (37°C)
To increase oxygen levels small volumes (µl) of 200 mM H2O2 stock concentration are injected into the O2k-chamber filled with MiR06.
The oxygen solubility factor of MiR06 is the same as for MiR05 = 0.92.
Preparation of MiR05 (MiR06) stock solution
- 1) weigh given amounts of the listed chemicals (except BSA and lactobionic acid) and transfer to a 1000 ml glass beaker
- 2) disrupt big lumps mechanically. It is recommended to do this before adding water, because during dissolution these lumps do not disintegrate easily.
- 3) add ~800 ml H2O and dissolve on a magnetic stirrer at ~30 °C
- 4) add 120 ml of K-lactobionate stock solution
- 5) adjust the pH to 7.1 with 5 N KOH at 30 °C
- 6) transfer the MiR05 stock solution to a 1000 ml volumentric glass flask and add H20 to a final volume of 1000 ml = MiR05
- 7) check pH again and adjust if necessary with small amounts of 5 N KOH
- 8) 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)
- 9) to prepare MiR06, add 280 000 units of catalase (100 mg of catalase powder containing 2800 u/mg solid) per 1 L MiR05 (280 units / ml final concentration)
- 10) divide into 40 ml portions and store at -20 °C in plastic vials
- MiR06 can also be prepared by adding 5 µl of the 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 Oxygraph-2k chamber = 280 u/ml.
Chemicals for MiR05/MiR06
Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
Compound | Final conc. | FW | Addition to 1 litre final volume | Company, product code and storage |
---|---|---|---|---|
EGTA | 0.5 mM | 380.4 | 0.190 g | Sigma E 4378, 25 g, store at R.T. |
MgCl2.6H20 | 3 mM | 203.3 | 0.610 g | Scharlau MA 0036, 250 g, store at R.T. |
Taurine | 20 mM | 125.1 | 2.502 g | Sigma T 0625, 25 g, store at R.T. |
KH2PO4 | 10 mM | 136.1 | 1.361 g | Merck 104873, 1000 g, store at R.T. |
HEPES | 20 mM | 238.3 | 4.77 g | Sigma H 7523, 250 g, store at R.T. |
D-Sucrose | 110 mM | 342.3 | 37.65 g | Roth, 4621.1, 1000 g, store at R.T. |
BSA essentially fatty acid free | 1 g/l | 1 g | Sigma A 6003 fraction V, 25 g, store at 2-8 °C | |
Lactobionic acid | 60 mM | 358.3 | 120 ml of 0.5 M K-lactobionate stock | Aldrich 153516 or Sigma L2398, 100 g, store at R.T. |
Preparation of K-lactobionate stock solution
- 1) weigh 35.83 g lactobionic acid into a 250 ml glass beaker
- 2) add 100 ml H2O and dissolve by stirring on magnetic stirrer
- 2) check pH (is approx. 2.0) and neutralize with 5 N KOH
- 4) adjust final volume to 200 ml with H2O. It is best to use a 200 ml volumetric glass flask.
- 5) check pH again and adjust to 7 if necessary (5 N KOH)
Preparation of catalase stock solution
- Catalase lypophilized powder, 2000-5000 units*/mg, Sigma C 9322, store at -20 °C
- Stock solution: 112000 u/ml (dissolved in MiR05)
- Example: 'Catalase lypophilized powder, 2800 units/mg solid and 3500 units/mg protein'
- 1) Use 'units/mg solid' for your calculations
- 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.
- 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.
Unit definition: * Units of enzymatic activitiy (u) in µmol/min; assay used by Sigma Aldrich: ' One unit will decompose 1.0 μmole of H2O2 per min at pH 7.0 at 25 °C, while the H2O2 concentration falls from 10.3 to 9.2 mM, measured by the rate of decrease of A240. '
Preparation of 200 mM H2O2 stock solution
H2O2: Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge, please see e.g. this link for handling and savety instructions concerning hydrogen peroxide.
- 1) pipette 114 µl of 17.6 M H2O2 into 10 ml plastic vial
- 2) add H2O to a total volume of 10 ml
- 3) wrap plastic vial in aluminium foil (solution is light sensitive) and store in the fridge
- 4) during experiments keep the solution on ice
titration of 3 µl of H2O2 into 2 ml chamber would increase the concentration of O2 by approx. 150 nmol/ml
For detailed information on chemicals see MiPNet14.13.
Preparation of MiR07
- 1) prepare every morning fresh by adding 3 mg/mL creatine monohydrate (Fluka 27900) to MiR06
- 2) stirr gently on a magnetic stirrer
Limitations of using MiR05 to MiR07
The intracellular milieu of kidney has a low [K+]. Kidney mitochondria are inhibited by the high [K+] of MiR05 to MiR07.
Reference on a mitochondrial respiration medium for kidney: Persson_2012_Diabetologia.
Info
MiPNet10.11 is integrated in MiPNet14.13_Medium-MiR06.