Reference: A: short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) -SUIT-006
SUIT number: D034_1PM;2D;3Omy;4U;5Ama
SUIT-006 Fluo mt D034 is a protocol to investigate the O2 flux and mitochondrial membrane potential with fluorescent dyes. In this protocol, the NADH Electron transfer-pathway state can be analysed mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. Addition of PM (pyruvate & malate) to the mitochondria leads to the hyperpolarisation of the mt-membrane, while ADP (D) decreases the mt-membrane potential. Addition of oligomycin (Omy) results in hyperpolarisation since the inhibition of the ATP synthase leads to an accumulation of protons in the intermembrane space. Uncoupler depolarises the mt-membrane in a concentration-dependent manner and antimycin A blocks the respiration and dissipates the mt-membrane potential. Since high concentrations of Omy can decrease the ET-capacity induced by addition of uncoupler, the optimal concentration of Omy has to be determined.
Communicated by Komlodi T, Iglesias-Gonzalez J and Gnaiger E (last update 2019-04-02)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
- » Selected keywords in SUIT protocols
- Pathway control
Strengths and limitations
- Before performing this protocol, a calibration with the fluorescence dye needs to be done. More information on our USB: Instrumental Protocols/Fluo calibration.
- It is recommended to run a chemical background without any sample to test the effect of the chemicals on the fluorescence signal.
- - Many fluorescence dyes (such as Safranin, TMRM, Rhodamine 123, etc) can inhibit components of the ETS, most commonly affecting NADH-linked respiration. Therefore, an experimental control should be done in the absence of the fluorescence dye to check for inhibitory effects. The following protocol is suggested: SUIT-006 O2 mt D047.
- - CIV activity cannot be determined and cytochrome c test cannot be performed together with the fluorescence dyes.
- - Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.
Compare SUIT protocols
- SUIT-020 Fluo mt D033: this is an expanded protocol to study mt-membrane potential not only with PM but also with glutamate and succinate to support NS(PGM)-pathway.
- SUIT-020 Fluo mt D036: this is a similar protocol to SUIT-020 Fluo mt D033 for the samples which display a preference for GM instead of PM.
|MiPNet20.13 Safranin mt-membranepotential||2018-09-25||Mouse||Nervous system|
|Krumschnabel 2014 Methods Enzymol||2014||Mouse||Nervous system|
MitoPedia methods: Fluorometry