During cellular respiration, nutrients are oxidized to generate energy through a mechanism called oxidative phosphorylation, which occurs in the mitochondria. During oxidative phosphorylation, the gradual degradation of molecules through the TCA cycle releases electrons from the covalent bonds that are broken. These electrons are captured by NAD+ through its reduction into NADH. Finally, NADH transports the electrons to the complexes of the electron chain in the internal membrane of mitochondria. These complexes use the energy released by the electrons to pump protons into the intermembrane space, generating an electrochemical gradient across the internal membrane of mitochondria, which provides energy for the ATP-synthase complex, ultimately producing adenosine triphosphate (ATP). We assessed the mitochondrial membrane potential (ψm) using tetramethylrhodamine methyl ester (TMRM), a cell-permeant, cationic, red fluorescent dye. To measure specifically the mitochondrial membrane potential (ψm) we quantified the fluorescence intensity before and after applying FCCP, a mitochondrial electron chain uncoupler. The difference of intensity before and after applying FCCP corresponds specifically to the mitochondrial membrane potential. We analyzed mitochondrial membrane potential (ψm) by cytofluorimetry. The ratio between the total level of signal and the signal generated after uncoupling provided a normalized value for the difference in cell size. Furthermore, to normalize for the different size of cells that we were analyzing we have analyzed TMRM in live imaging using IN Cell Analyzer, so that the level of mitochondrial membrane potential could be detected per unit of mitochondrial membrane area measured. Thus, our protocol can also be used to compare the mitochondrial membrane potential of cells that are different in size.
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