MiPNet14.13 Medium-MiR06

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Mitochondrial respiration medium - MiR06.

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Oroboros (2016-08-30) Mitochondr Physiol Network

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 





HRR: O2k-Protocol 

O2k-chemicals and media 

Application of MiR06 in HRR

MiR06: Mitochondrial Respiration Medium (MiR06 = MiR05 + Catalase).

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)


Re-oxygenation

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

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. '

MiR05Cr/MiR06Cr

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 H2O2 stock solutions

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.


Preparation of 200 mM stock solution (dissolved in H2O) for 2-mL O2k-chamber:
  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 aluminum foil (solution is light-sensitive) and store at 4 °C.
  4. During experiments keep the stock solution on ice.
»O2k manual titrations: MiPNet09.12
  • Titration volume (2-mL O2k-chamber): 1-3 µL using a 10 µL Hamilton syringe.
  • 3 µL of H2O2 into the 2-mL O2k-chamber increases the concentration of O2 by approx. 150 nmol/mL (150 µM).


Preparation of 50 mM stock solution (dissolved in H2O) for 0.5-mL O2k-chamber:
  1. Pipette 28 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 aluminum foil (solution is light-sensitive) and store at 4 °C.
  4. During experiments keep the stock solution on ice.
»O2k manual titrations: MiPNet09.12
  • Titration volume (0.5-mL O2k-chamber): 1-3 µL using a 10 µL Hamilton syringe.
  • 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 [1].

>> MiPMap - Is this a general issue for the organ, or is it in addition also a species issue?

  1. A mitochondrial respiration medium for kidney: Friederich-Persson 2012 Diabetologia.

Further information

  • MiPNet08.05 and MiPNet10.11 are integrated in MiPNet14.13_Medium-MiR06.

Original publication introducing MiR05: