Difference between revisions of "MiPNet03.02 Chemicals-Media"
From Bioblast
| Line 1: | Line 1: | ||
{{Publication | {{Publication | ||
|title=[[Image:O2k-Protocols.jpg|right|80px|link= | |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] Selected media and chemicals for respirometry with mitochondrial preparations. [[Media:MiPNet03.02 Chemicals-Media.pdf |»Bioblast pdf«]] | ||
|info=[http://www.bioblast.at/index.php/File:MiPNet03.02_Chemicals-Media.pdf Versions] | |info=[http://www.bioblast.at/index.php/File:MiPNet03.02_Chemicals-Media.pdf Versions] | ||
|authors=OROBOROS | |authors=OROBOROS | ||
| Line 8: | Line 8: | ||
Different media for tissue preparation and respiration are used in investigations of mitochondrial function. Initial decisions on the composition of media and chemicals are decisive for long-term studies and crucial for comparability of results. As a guideline, we summarize an update of our experience with media and chemicals for high-resolution respirometry with isolated mitochondria, permeabilized cells, muscle fibres and tissue homogenates. Whereas optimization is necessary for specific experimental protocols, standardization will improve the comparability of results obtained in different laboratories. Efforts towards standardization are important for the advancement of mitochondrial physiology. | Different media for tissue preparation and respiration are used in investigations of mitochondrial function. Initial decisions on the composition of media and chemicals are decisive for long-term studies and crucial for comparability of results. As a guideline, we summarize an update of our experience with media and chemicals for high-resolution respirometry with isolated mitochondria, permeabilized cells, muscle fibres and tissue homogenates. Whereas optimization is necessary for specific experimental protocols, standardization will improve the comparability of results obtained in different laboratories. Efforts towards standardization are important for the advancement of mitochondrial physiology. | ||
: | :» Product: [[OROBOROS O2k]], [[O2k-Catalogue_OROBOROS | O2k-Catalogue]] | ||
|mipnetlab=AT Innsbruck OROBOROS}} | |mipnetlab=AT Innsbruck OROBOROS}} | ||
{{Labeling | {{Labeling | ||
| Line 15: | Line 15: | ||
|additional=O2k-chemicals and media}} | |additional=O2k-chemicals and media}} | ||
== Supplementary information == | == Supplementary information == | ||
: | :» [[MiPNet09.12 O2k-Titrations]]; [[MitoPedia]] | ||
: | :» [http://www.oroboros.at/?CytochromecControl Cytochrome ''c'' control] | ||
: | :» For calculations, see Excel file: [http://www.oroboros.at/?Protocols_titrations O2k-Titrations.xls] | ||
: | :» For pH adjustment of BIOPS solution, note the temperature dependence: | ||
:::::::* at 0 °C pH = 7.1 | :::::::* at 0 °C pH = 7.1 | ||
:::::::* at 23 °C pH = 6.75 | :::::::* at 23 °C pH = 6.75 | ||
| Line 24: | Line 24: | ||
== History == | == History == | ||
* Malate was listed for a final concentration of 2 mM until 2013. During this year, test exeriments with mitochondria from various tissues and species and with different mt-preparations (isolated mitochondria, permeabilized fibres, tissue homogenate) revealed an inhibitory effect of 2 mM malate on CII linked respiration (S | * Malate was listed for a final concentration of 2 mM until 2013. During this year, test exeriments with mitochondria from various tissues and species and with different mt-preparations (isolated mitochondria, permeabilized fibres, tissue homogenate) revealed an inhibitory effect of 2 mM malate on CII linked respiration (S,Rot). The inhibitory effect is less at 0.5 mM malate, and 0.5 mM malate may be saturating for CI-linked respiration. Evaluation is required for the optimum malate concentrations in SUIT protocols with sequential CI, CI<small>&</small>II and CII substrate states. | ||
* [[Carbonyl cyanide m-chloro phenyl hydrazone|From FCCP to CCCP]] | * [[Carbonyl cyanide m-chloro phenyl hydrazone|From FCCP to CCCP]] | ||
* Up to 2006, rotenone was added at a high final concentration (0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggested to use a stock solution at lower concentration (0.1 mM), to reduce the problem of rotenone retention. The 2006 edition listed a 0.2 mM stock solution, but the amount added to 10 ml ethanol was given for a 0.15 mM stock solution. Our recent rotenone titrations with permeabilized muscle fibers, however, confirmed that the originally proposed higher rotenone concentration is actually required for full inhibition. | * Up to 2006, rotenone was added at a high final concentration (0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggested to use a stock solution at lower concentration (0.1 mM), to reduce the problem of rotenone retention. The 2006 edition listed a 0.2 mM stock solution, but the amount added to 10 ml ethanol was given for a 0.15 mM stock solution. Our recent rotenone titrations with permeabilized muscle fibers, however, confirmed that the originally proposed higher rotenone concentration is actually required for full inhibition. | ||
Revision as of 05:17, 23 June 2015
 |
» Versions
OROBOROS (2015-03-30) Mitochondr Physiol Network
Abstract: Fontana-Ayoub M, Fasching M, Gnaiger E (2014) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(17):1-9.
Different media for tissue preparation and respiration are used in investigations of mitochondrial function. Initial decisions on the composition of media and chemicals are decisive for long-term studies and crucial for comparability of results. As a guideline, we summarize an update of our experience with media and chemicals for high-resolution respirometry with isolated mitochondria, permeabilized cells, muscle fibres and tissue homogenates. Whereas optimization is necessary for specific experimental protocols, standardization will improve the comparability of results obtained in different laboratories. Efforts towards standardization are important for the advancement of mitochondrial physiology.
- » Product: OROBOROS O2k, O2k-Catalogue
• 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.
O2k-chemicals and media
Supplementary information
- » MiPNet09.12 O2k-Titrations; MitoPedia
- » Cytochrome c control
- » For calculations, see Excel file: O2k-Titrations.xls
- » For pH adjustment of BIOPS solution, note the temperature dependence:
- at 0 °C pH = 7.1
- at 23 °C pH = 6.75
History
- Malate was listed for a final concentration of 2 mM until 2013. During this year, test exeriments with mitochondria from various tissues and species and with different mt-preparations (isolated mitochondria, permeabilized fibres, tissue homogenate) revealed an inhibitory effect of 2 mM malate on CII linked respiration (S,Rot). The inhibitory effect is less at 0.5 mM malate, and 0.5 mM malate may be saturating for CI-linked respiration. Evaluation is required for the optimum malate concentrations in SUIT protocols with sequential CI, CI&II and CII substrate states.
- Up to 2006, rotenone was added at a high final concentration (0.5 µM), hence a 0.5 mM stock solution was prepared. Since 0.05 µM may be fully inhibiting, we suggested to use a stock solution at lower concentration (0.1 mM), to reduce the problem of rotenone retention. The 2006 edition listed a 0.2 mM stock solution, but the amount added to 10 ml ethanol was given for a 0.15 mM stock solution. Our recent rotenone titrations with permeabilized muscle fibers, however, confirmed that the originally proposed higher rotenone concentration is actually required for full inhibition.
