Adaptation of the technique of determination of mitochondrial membrane potential change in C2C12 cells using the ready set “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich)

Abstract

   Background. We used the “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich) to detected MMP but encountered difficulties by applied this kit because in manual there not JC-10 concentration and it not allowing for account cell type, size, density, differences in incubation time for different cell cultures.   Objective. Adaptation of the method for determining MMP (mitochondrial membrane potential) in C2C12 cells using in microplate reader and electron microscope.   Design and methods. MMP in C2C12 cells was measured by two way, using fluorescence microscopy (Zeiss, Zen program) and using a plate fluorimeter (CLARIOstar (BMG LABTECH). JC-10 and TMRE dyes (Sigma-Aldrich) were used as fluorescent probes.   Results. Optimal conditions for detection changes in mitochondrial membrane potential in C2C12 cells were selected. 100- fold dilution of the dye JC-10 (Dye Loading Solution) and replacement of the manufactured buffer to PBS led to repeatability and reproducibility results.   Conclusion. When using ready-made kits for measuring MMP, the method proposed by the manufacturer may not be suitable for the selected cell line. In our study to mouse myoblasts of the C2C12 line, a dilution of the dye for loading was required 100 times compared to that recommended by the manufacturer.