MiPNet14.13 Medium-MiR06: Difference between revisions
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{{OROBOROS header page name}} | |||
{{Publication | {{Publication | ||
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Mitochondrial respiration medium - MiR06. [[ | |||
|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols]] Mitochondrial respiration medium - MiR06. | |||
|authors= | |info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/d/d9/MiPNet14.13_Medium-MiR06.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet14.13_Medium-MiR06.pdf Versions] | ||
|year= | |authors=Oroboros | ||
|year=2016-08-30 | |||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|abstract=Fasching M, Fontana-Ayoub M, Gnaiger E ( | |abstract='''Fasching M, Fontana-Ayoub M, Gnaiger E (2018) Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13(06):1-4.''' | ||
{{MiPNet pdf page linking to MitoPedia}} | |||
Mitochondrial respiration medium MiR06 was developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06+creatine. | Mitochondrial respiration medium MiR06 was developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06+creatine. | ||
: | |||
:» Product: [[MiR05-Kit]] | |||
|keywords=MiR06 | |keywords=MiR06 | ||
|mipnetlab= | |mipnetlab=AT_Innsbruck_Oroboros | ||
}} | |||
{{Labeling | {{Labeling | ||
|area=Instruments;methods | |area=Instruments;methods | ||
|instruments=Protocol | |instruments=O2k-Protocol | ||
|additional=O2k-chemicals and media}} | |additional=O2k-chemicals and media | ||
}} | |||
__TOC__ | |||
== Application of MiR06 in [[HRR]] == | == Application of MiR06 in [[HRR]] == | ||
Line 24: | Line 32: | ||
== Re-oxygenation | == Re-oxygenation == | ||
An experiment needs not necessarily be terminated , because of running out of oxygen. There are different | ::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 == | == Preparation of MiR05 (MiR06) stock solution == | ||
* Total volume of [[solution]] = 1 litre. | * Total volume of [[solution]] = 1 litre. | ||
::1) Weigh given amounts of the [[ | ::1) Weigh given amounts of the [[Media:MiPNet14.13 Medium-MiR06.pdf| 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. | ::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 | ::3) Add ~800 mL H<sub>2</sub>O and dissolve using a magnetic stirrer at ~30 °C | ||
::4) Add 120 | ::4) Add 120 mL of [[#Preparation of K-lactobionate stock solution|K-lactobionate stock solution]]. | ||
::5) Adjust the pH to 7.1 with 5 | ::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). | ::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 H<sub>2</sub>0 to a final volume of 1000 | ::7) Add H<sub>2</sub>0 to a final volume of 1000 mL. | ||
::8) Check pH again and adjust if necessary with small volumes of 5 | ::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 <span style="color:#2E8B57"> '''MiR06'''</span>, add 280 000 units of catalase (100 mg of catalase powder containing 2800 | ::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 litre MiR05 (280 units / mL final concentration). | ||
::10) Divide into 40 | ::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 [[solution]]s. 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 | ::11) These storage solutions of MiR06 can be used as stock [[solution]]s. 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. | ||
::<span style="color:#2E8B57"> '''MiR06''' </span> can also be prepared by adding 5 | ::<span style="color:#2E8B57"> '''MiR06''' </span> can also be prepared by adding 5 µL 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 O2k chamber is 280 U/mL. | ||
=== Preparation of K-lactobionate stock solution === | === Preparation of K-lactobionate stock solution === | ||
::1) weigh 35.83 g lactobionic acid into a 250 | ::1) weigh 35.83 g lactobionic acid into a 250 mL glass beaker | ||
::2) add 100 | ::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 | ::2) check pH (is approx. 2.0) and neutralize with 5 M KOH | ||
::4) adjust final volume to 200 | ::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 | ::5) check pH again and adjust to 7 if necessary (5 M KOH) | ||
=== Preparation of catalase stock solution === | === Preparation of catalase stock solution === | ||
::'''Catalase''' lypophilized powder, 2000-5000 ''' | ::'''Catalase''' lypophilized powder, 2000-5000 '''Units*'''/mg, Sigma C 9322, store at -20 °C | ||
::'''Stock solution:''' 112000 | ::'''Stock solution:''' 112000 U/mL (dissolved in MiR05) | ||
::'''Example:''' 'Catalase lypophilized powder, 2800 | ::'''Example:''' 'Catalase lypophilized powder, 2800 Units/mg solid and 3500 Units/mg protein' | ||
::1) Use ' | ::1) Use 'Units/mg solid' for your calculations | ||
::2) Result: 40 mg catalase powder (2800 | ::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 | ::3) Titrate 5 µL of the catalase stock solution into the 2 mL chamber to achieve a final concentration of 280 U/mL in the chamber. | ||
'''Unit definition:''' '''* Units''' of enzymatic activitiy ( | '''Unit definition:''' '''* Units''' of enzymatic activitiy (U) in µmol/min; assay used by Sigma Aldrich: ' ''One unit will decompose 1.0 μmol of H<sub>2</sub>O<sub>2</sub> per min at pH 7.0 at 25 °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>.'' ' | ||
== MiR05Cr/MiR06Cr == | == MiR05Cr/MiR06Cr == | ||
Line 85: | Line 79: | ||
* [[MiR06Cr]] = MiR06 + Creatine | * [[MiR06Cr]] = MiR06 + Creatine | ||
::1) Prepare fresh by adding 3 mg/ | ::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. | ::2) Stirr gently on a magnetic stirrer. | ||
::3) Do not freeze to avoid precipitation. | ::3) Do not freeze to avoid precipitation. | ||
== Preparation of | == Preparation of H<sub>2</sub>O<sub>2</sub> stock solutions == | ||
'''H<sub>2</sub>O<sub>2</sub>''': Hydrogen peroxide solution, 50 wt. % in | :::: '''H<sub>2</sub>O<sub>2</sub>''': Hydrogen peroxide solution, 50 wt. % in H<sub>2</sub>O, stabilized, Sigma 516813, store in the fridge. See [http://www.h2o2.com/technical-library/default.aspx?pid=66&name=Safety-amp-Handling this link] for handling and safety instructions concerning hydrogen peroxide. | ||
''' | :::: '''Preparation of 200 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''2-mL O2k-chamber''': | ||
::::# Pipette 114 µL of 17.6 M H<sub>2</sub>O<sub>2</sub> into 10 mL plastic vial. | |||
::::# Add H<sub>2</sub>O, acidify with HCl (1 mM) to pH 6, complete with H<sub>2</sub>O to a total volume of 10 mL. Maintain the pH in the stock solution acidic to minimize autoxidation. | |||
::::# Wrap plastic vial in aluminum foil (solution is light-sensitive) and store at 4 °C. | |||
::::# During experiments keep the stock solution on ice. | |||
:::»'''O2k manual titrations:''' [[MiPNet09.12]] | |||
* MiR06 or MiR06Cr cannot be used for measurement of ROS production. Use MiR05 or MiR05Cr instead. | ::::* Titration volume ('''2-mL O2k-chamber'''): 1-3 µL using a 10 µL Hamilton syringe. | ||
* The high antioxidant activity may compete with reactions on which measurement of ROS production is based. | ::::* 3 µL of H<sub>2</sub>O<sub>2</sub> into the 2-mL O2k-chamber increases the concentration of O<sub>2</sub> by approx. 150 nmol/mL (150 µM). | ||
* 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]. | |||
:::: '''Preparation of 50 mM stock solution''' (dissolved in H<sub>2</sub>O) for '''0.5-mL O2k-chamber''': | |||
::::# Pipette 28 µL of 17.6 M H<sub>2</sub>O<sub>2</sub> into 10 mL plastic vial. | |||
::::# Add H<sub>2</sub>O, acidify with HCl (1 mM) to pH 6, complete with H<sub>2</sub>O to a total volume of 10 mL. Maintain the pH in the stock solution acidic to minimize autoxidation. | |||
::::# Wrap plastic vial in aluminum foil (solution is light-sensitive) and store at 4 °C. | |||
::::# 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 H<sub>2</sub>O<sub>2</sub> into the 2-mL O2k-chamber increases the concentration of O<sub>2</sub> by approx. 150 nmol/mL (150 µM). | |||
<!-- | |||
'''Titration''' of 3 µL of H<sub>2</sub>O<sub>2</sub> into the 2-mL O2k-chamber increases the concentration of O<sub>2</sub> 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<sup>+</sup>]. Kidney mitochondria are inhibited by the high [K<sup>+</sup>] of MiR05 to MiR06Cr [1]. | |||
>> [[MiPMap#1._Human_and_model_organisms.2C_taxonomic_groups|MiPMap - Is this a general issue for the organ, or is it in addition also a species issue?]] | >> [[MiPMap#1._Human_and_model_organisms.2C_taxonomic_groups|MiPMap - Is this a general issue for the organ, or is it in addition also a species issue?]] | ||
# A mitochondrial respiration medium for kidney: [[ | # A mitochondrial respiration medium for kidney: [[Friederich-Persson 2012 Diabetologia]]. | ||
== Further information == | == Further information == | ||
: | ::* [[MiPNet06.03_O2-Calibration-Solubility |Oxygen solubility in MiR06]] | ||
: | ::* [[MitoPedia: Media for respirometry]] | ||
: | ::* MiPNet08.05 and MiPNet10.11 are integrated in MiPNet14.13_Medium-MiR06. | ||
Original publication introducing MiR05: | '''Original publication introducing MiR05:''' | ||
* [[Gnaiger 2000 Life in the Cold|Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York: pp 431-442.]] | * [[Gnaiger 2000 Life in the Cold|Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York: pp 431-442.]] |
Latest revision as of 10:25, 16 March 2022
MiPNet14.13 Medium-MiR06
Mitochondrial respiration medium - MiR06. |
» »Versions
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.
- » Current O2k-series: NextGen-O2k Series XB and O2k Series J
- » Current software versions DatLab 8.0: MitoPedia: DatLab
- Further details: » MitoPedia: O2k-Open Support
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
- 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 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 1000 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 112000 U/mL.
- 3) Titrate 5 µL of the catalase stock solution into the 2 mL chamber to achieve a final concentration of 280 U/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:
- Pipette 114 µL of 17.6 M H2O2 into 10 mL plastic vial.
- 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.
- Wrap plastic vial in aluminum foil (solution is light-sensitive) and store at 4 °C.
- 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:
- Pipette 28 µL of 17.6 M H2O2 into 10 mL plastic vial.
- 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.
- Wrap plastic vial in aluminum foil (solution is light-sensitive) and store at 4 °C.
- 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?
- 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: