Abstract: Fasching M, Fontana-Ayoub M, Gnaiger E (2018) Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13(06):1-4.
Mitochondrial respiration medium MiR06 was developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06+creatine.
- » Product: MiR05-Kit
• Keywords: MiR06
• O2k-Network Lab: AT_Innsbruck_Oroboros
Labels: MiParea: Instruments;methods
O2k-chemicals and media
- 1 Application of MiR06 in HRR
- 2 Re-oxygenation
- 3 Preparation of MiR05 (MiR06) stock solution
- 4 MiR05Cr/MiR06Cr
- 5 Preparation of 200 mM H2O2 stock solution
- 6 Limitations of using MiR media
- 7 Further information
Application of MiR06 in HRR
- Oxygen solubility factor in MiR05 or 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)
- An experiment needs not necessarily be terminated, because of running out of oxygen. There are different ways to re-oxygenate. See all of them in Setting the oxygen concentration
Preparation of MiR05 (MiR06) stock solution
- Total volume of solution = 1 litre.
- 1) Weigh given amounts of the listed chemicals (except BSA and lactobionic acid) and transfer to a 1,000 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 using a magnetic stirrer at ~30 °C
- 4) Add 120 mL of K-lactobionate stock solution.
- 5) Adjust the pH to 7.1 with 5 M KOH at 30 °C.
- 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).
- 7) Add H20 to a final volume of 1,000 mL.
- 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.
- 9) To prepare MiR06, add 280 000 units of catalase (100 mg of catalase powder containing 2800 u/mg solid) per litre MiR05 (280 units / mL final concentration).
- 10) Divide into 40 mL portions in plastic vials and store at -20 °C.
- 11) These storage solutions of MiR06 can be used as stock solutions. 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.
- 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 O2k chamber is 280 u/mL.
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 M 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 M 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 112,000 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 μmol 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. '
- 1) Prepare fresh by adding 3 mg/mL creatine monohydrate (Fluka 27900, 100 g) to MiR05 or MiR06.
- 2) Stirr gently on a magnetic stirrer.
- 3) Do not freeze to avoid precipitation.
Preparation of 200 mM H2O2 stock solution
H2O2: Hydrogen peroxide solution, 50 wt. % in H2O, stabilized, Sigma 516813, store in the fridge. See this link for handling and safety instructions concerning hydrogen peroxide.
- 1) Pipette 114 µL of 17.6 M H2O2 into 10 mL plastic vial.
- 2) Add H2O, 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.
- 3) Wrap plastic vial in aluminium foil (solution is light sensitive) and store at 4 °C.
- 4) During experiments keep the stock solution on ice.
Titration of 3 µL of H2O2 into the 2 mL O2k-chamber increases the concentration of O2 by approx. 150 nmol/mL (150 µM).
Limitations of using MiR media
- MiR06 or MiR06Cr cannot be used for measurement of ROS production. Use MiR05 or MiR05Cr instead.
- The high antioxidant activity may compete with reactions on which measurement of ROS production is based.
- The intracellular milieu of kidney has a low [K+]. Kidney mitochondria are inhibited by the high [K+] of MiR05 to MiR06Cr .
- A mitochondrial respiration medium for kidney: Friederich-Persson 2012 Diabetologia.
- MiPNet08.05 and MiPNet10.11 are integrated in MiPNet14.13_Medium-MiR06.
Original publication introducing MiR05: