Template:SUIT text D034

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 in 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 organisms 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.