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Difference between revisions of "MiPNet14.13 Medium-MiR06"

From Bioblast
Line 175: Line 175:
:>> [[List_of_media_for_respirometry|List of respiration media]]
:>> [[List_of_media_for_respirometry|List of respiration media]]


:>> [http://www.oroboros.at/?Protocols_MiR06 Protocols MiR06]
:>> MiPNet08.05 and MiPNet10.11 are integrated in MiPNet14.13_Medium-MiR06.
:>> MiPNet08.05 and MiPNet10.11 are integrated in MiPNet14.13_Medium-MiR06.



Revision as of 10:20, 2 December 2013

Publications in the MiPMap
Fasching M, Eigentler A, Fontana-Ayoub M, Gnaiger E (2013) Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13.

» Open Access-MiPNet14.13.pdf; Versions

O2k-Protocols contents

OROBOROS (2013) Mitochondr Physiol Network

Abstract: O2k-Protocol for Mitochondrial Respiration Medium - MiR06

Mitochondrial respiration medium MiR06 was developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06+creatine.

>> O2k-Protocols:Overall contents
>> Product: OROBOROS Oxygraph-2k, O2k-Catalogue

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 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

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 or Sigma P5655 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, or Sigma 84097, 1000 g, store at R.T., Note a
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.
  • Note a: For specific applications contamination with glucose may be critical. Invert sugar (1:1 mixture of glucose and fructose) is specified for Roth #4621 (< 0.04%), but not for Sigma #84097. Invert sugar content is specified for Sigma #84100 (<0.05%), however the overall specified purity for this product (>= 99%) is lower than for Sigma #8407 and Roth #4621.1 (both products >= 99.5%).

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

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. See 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 this 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 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.


Further information

>> Oxygen solubility in MiR06
>> List of respiration media
>> MiPNet08.05 and MiPNet10.11 are integrated in MiPNet14.13_Medium-MiR06.
  • Version 3: 2013-06-20

Original publication introducing MiR05: