SUIT-009 O2 mt D015: Difference between revisions
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== 1S;2D;3P;4Ama-== | == 1S;2D;3P;4Ama-== | ||
SUIT 9 is a short protocol for simultaneous determination of O2 flux and the rate of H<sub>2</sub>O<sub>2</sub> production (H<sub>2</sub>O<sub>2</sub> flux) on isolated mitochondria and permeabilized cells. Succinate (S) supports the reverse electron transport (RET) initiated H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Addition of ADP (D) decreases H<sub>2</sub>O<sub>2</sub> flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports NADH-linked (N) pathway, and usually in the presence of D it does not increase further the H<sub>2</sub>O<sub>2</sub> flux. Antimycin (Ama) inhibits Complex III (CIII) and results in the elevation of the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRedยฎ assay is changing over the experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes several H<sub>2</sub>O<sub>2</sub> calibration steps are | SUIT 9 is a short protocol for simultaneous determination of O2 flux and the rate of H<sub>2</sub>O<sub>2</sub> production (H<sub>2</sub>O<sub>2</sub> flux) on isolated mitochondria and permeabilized cells. Succinate (S) supports the reverse electron transport (RET) initiated H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Addition of ADP (D) decreases H<sub>2</sub>O<sub>2</sub> flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports NADH-linked (N) pathway, and usually in the presence of D it does not increase further the H<sub>2</sub>O<sub>2</sub> flux. Antimycin (Ama) inhibits Complex III (CIII) and results in the elevation of the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRedยฎ assay is changing over the experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes several H<sub>2</sub>O<sub>2</sub> calibration steps are done during the experiment. The measurement is carried out in MiR05 at 37oC. ย | ||
== SUIT O2 DLP for SUIT 9 (DatLab 7.1) == | == SUIT O2 DLP for SUIT 9 (DatLab 7.1) == |
Revision as of 10:25, 21 March 2018
Description
Abbreviation: NS(P)
Reference: B: SUIT 9 is a short SUIT protocol for H2O2 (AmR) in mitochondrial preparations.
SUIT number: 9
O2k-Application: AmR
MitoPedia concepts:
SUIT protocol,
SUIT B
- SUIT-category: NS(P)
1S;2D;3P;4Ama-
SUIT 9 is a short protocol for simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) on isolated mitochondria and permeabilized cells. Succinate (S) supports the reverse electron transport (RET) initiated H2O2 production in the LEAK state. Addition of ADP (D) decreases H2O2 flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports NADH-linked (N) pathway, and usually in the presence of D it does not increase further the H2O2 flux. Antimycin (Ama) inhibits Complex III (CIII) and results in the elevation of the H2O2 flux. The sensitivity of the Amplex UltraRedยฎ assay is changing over the experimental time and upon addition of chemicals. To correct H2O2 flux for the sensitivity changes several H2O2 calibration steps are done during the experiment. The measurement is carried out in MiR05 at 37oC.
SUIT O2 DLP for SUIT 9 (DatLab 7.1)
- DL-Protocols (DLP) for substrate-uncoupler-inhibitor titrations (see MitoPedia: SUIT) guide through a sequence of coupling and pathway control states. A specific SUIT protocol can be assigned to O2k-Chamber A or B or both. DL-Protocols for different SUIT protocols can be found in the library of protocols MitoPedia: SUIT. Instrumental DL-protocols are displayed in the DL-Protocol library.
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