MiPNet14.13 Medium-MiR06

Fasching M, Eigentler A, Fontana-Ayoub M, Gnaiger E (2009) Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13.

» MiPNet14.13 Open Access

OROBOROS (2009) Mitochondr Physiol Network

Abstract: Mitochondrial respiration medium MiR06 was developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. • Keywords: MiR06

• O2k-Network Lab: AT_Innsbruck_OROBOROS

Labels: MiParea: Instruments;methods 

HRR: 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. 

Chemicals & 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 with H₂O₂ titrations

• To increase oxygen levels small volumes (µl) of [[#Preparation of 200 mM H₂O₂ stock solution|200 mM H₂O₂ stock solution]] are injected into the O2k-chamber filled with 2 ml MiR06.

With MiR06 (or MiR06Cr), the medium in the O2k-chamber can be re-oxygenated very conveniently with H₂O₂ 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 H₂O₂, 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 H₂O₂ titrations into the closed chamber do not lead to gas bubble formation.

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 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 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 N 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 1000 ml.

8) Check pH again and adjust if necessary with small volumes of 5 N 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 Oxygraph-2k chamber is 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 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

• MiR05Cr = MiR05 + Creatine
• MiR06Cr = MiR06 + Creatine

1) Prepare fresh by adding 3 mg/ml creatine monohydrate (Fluka 27900) to MiR05 or MiR06.

2) Stirr gently on a magnetic stirrer.

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.

Limitations of using MiR05 to MiR06Cr

• 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: Persson_2012_Diabetologia.

Versions

• Version 3: 2013-06-20
• MiPNet10.11 is integrated in MiPNet14.13_Medium-MiR06.