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| {{Publication | | {{Publication |
| |title=Fasching M, Eigentler A, Fontana-Ayoub M, Gnaiger E. Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13. | | |title=Fasching M, Renner K, Gnaiger E. Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13. |
| |info=[http://www.oroboros.at/?Protocols_MiR06 MiPNet14.13 Open Access] | | |info=[http://www.oroboros.at/index.php?protocols_miro6 MiPNet14.13] |
| |authors=Fasching M, Eigentler A, Fontana-Ayoub M, Gnaiger E | | |authors=Fasching M, Renner K, Gnaiger E |
| |year=* | | |year=* |
| |journal=Mitochondr Physiol Network | | |journal=Mitochondr Physiol Network |
| |abstract=Mitochondrial respiration medium 06, developed for oxygraph incubations of mitochondrial preparations = MiR05 plus catalase. | | |abstract=Mitochondrial respiration medium 06, developed for oxygraph incubations of mitochondrial preparations = MiR05 plus catalase. |
| | |
| | See: [[MiR06|Preparation of MiR06]] |
| |mipnetlab=AT_Innsbruck_OROBOROS | | |mipnetlab=AT_Innsbruck_OROBOROS |
| |articletype=Protocol; Manual, MiPNet-online Publication | | |articletype=Protocol; Manual, MiPNet-online Publication |
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| |discipline=Mitochondrial Physiology | | |discipline=Mitochondrial Physiology |
| }} | | }} |
| == Application of MiR06 in [[HRR]] == | | == Info == |
| Β | |
| '''MiR06: Mitochondrial Respiration Medium''' ([[#Preparation of MiR05 (MiR06) stock solution|MiR06]] = [[MiR05]] + [[Catalase]]). Total volume = 1 litre.
| |
| Β | |
| ::Oxygen solubility factor in MiR05 or MiR06 at 30Β°C and 37Β°C = 0.92
| |
| Β | |
| ::pH of MiR05/06: 7.2 (20Β°C), 7.2 (25Β°C), 7.1 (30Β°C), 7.1 (35Β°C), 7.0 (37Β°C)
| |
| Β | |
| Β | |
| == Re-oxygenation with H2O2 titrations ==
| |
| Β | |
| * '''To increase oxygen levels''' small volumes (Β΅l) 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.
| |
| Β | |
| With MiR06 (or [[MiR06Cr]]), the medium in the O2k-chamber can be re-oxygenated very conveniently with H<sub>2</sub>O<sub>2</sub> 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 availableΒ for initial oxygenation, a very small bubble may be left in the chamber while slowly rising the oxygen level to 500 Β΅M with additions of H<sub>2</sub>O<sub>2</sub>, 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 H<sub>2</sub>O<sub>2</sub> into the closed chamber do not lead to gas bubble formation.
| |
| Β | |
| Β | |
| == Preparation of MiR05 (MiR06) stock solution ==
| |
| Β | |
| ::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 Β°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 Β°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 Β°C in plastic vials
| |
| Β | |
| ::<span style="color:#2E8B57"> '''MiR06''' </span> can also be prepared by adding 5 Β΅l 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.
| |
| Β | |
| Β | |
| ==== Chemicals for MiR05/MiR06 ====
| |
| Β | |
| <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
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| |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 Β°C
| |
| |-
| |
| |align="right"|Lactobionic acid
| |
| |align="right"|60 mM
| |
| |align="right"|358.3
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| |align="right"|120 ml of 0.5 M [[#Preparation of K-lactobionate stock solution|K-lactobionate stock]]
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| |align="right"|Aldrich 153516 or Sigma L2398, 100 g, store at R.T.
| |
| |-
| |
| |}
| |
| Β | |
|
| |
| ==== Preparation of K-lactobionate stock solution ====
| |
| Β | |
| ::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)
| |
| Β | |
| Β | |
| === Preparation of catalase stock solution ===
| |
| Β | |
| ::'''Catalase''' lypophilized powder, 2000-5000 '''units*'''/mg, Sigma C 9322, store at -20 Β°C
| |
| Β | |
| ::'''Stock solution:''' 112000 u/ml (dissolved in MiR05)
| |
| Β | |
| Β | |
| ::'''Example:''' 'Catalase lypophilized powder, 2800 units/mg solid and 3500 units/mg protein'
| |
| Β | |
| ::1) Use 'units/mg solid' for your calculations
| |
| ::2) Result:Β 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 Β΅l of the catalase stock solution into the 2 ml chamber to achieve a final concentration of 280 IU/ml in the chamber.
| |
| Β | |
| '''Unit definition:''' '''* Units''' of enzymatic activitiy (u) in Β΅mol/min; assay used by Sigma Aldrich: ' ''One unit will decompose 1.0 ΞΌmole of H<sub>2</sub>O<sub>2</sub>Β per min at pH 7.0 at 25 Β°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>.'' '
| |
| Β | |
| Β | |
| === Preparation of 200 mM H2O2 stock solution ===
| |
| Β | |
| '''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.pdfΒ this link] for handling and savety instructions concerning hydrogen peroxide.
| |
| Β | |
| ::1) pipette 114 Β΅l 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
| |
| Β | |
| '''titration''' of 3 Β΅l 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
| |
| Β | |
| '''For detailed information on chemicals see [http://www.oroboros.at/index.php?protocols_miro6 MiPNet14.13].'''
| |
|
| |
|
| == MiR06Cr ==
| | MiPNet10.11 is integrated in MiPNet14.13_Medium-MiR06 |
|
| |
|
| * [[MiR06Cr]] = MiR06 + Creatine
| |
|
| |
|
| ::1) prepare every morning fresh by adding 3 mg/mL creatine monohydrate (Fluka 27900) to [[MiR06]]
| | == MiR07 == |
| ::2) stirr gently on a magnetic stirrer
| |
|
| |
|
| == Limitations of using MiR05 to MiR06Cr == | | MiR07 = MiR06 + 20 mM creatine |
|
| |
|
| * The high antioxidant activity may interfere with the measurement of ROS production.
| |
| * The intracellular milieu of kidney has a low [K<sup>+</sup>]. Kidney mitochondria are inhibited by the high [K<sup>+</sup>] of MiR05 to MiR06Cr [1].
| |
|
| |
|
| # Reference on a mitochondrial respiration medium for kidney: [[Persson_2012_Diabetologia]].
| | == Limitations of using MiR05 to MiR07 == |
|
| |
|
| | The intracellular milieu of kidney has a low [K<sup>+</sup>]. Kidney mitochondria are inhibited by the high [K<sup>+</sup>] of MiR05 to MiR07. |
|
| |
|
| MiPNet10.11 is integrated in MiPNet14.13_Medium-MiR06.
| | Reference on a mitochondrial respiration medium for kidney: [[Persson_2012_Diabetologia]]. |
Fasching M, Renner K, Gnaiger E. Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13.
|
Β» MiPNet14.13
Fasching M, Renner K, Gnaiger E (
) Mitochondr Physiol Network
Abstract: Mitochondrial respiration medium 06, developed for oxygraph incubations of mitochondrial preparations = MiR05 plus catalase.
See: Preparation of MiR06
β’ 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
Info
MiPNet10.11 is integrated in MiPNet14.13_Medium-MiR06
MiR07
MiR07 = MiR06 + 20 mM creatine
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.