A new method for cytofluorometric analysis of mitochondrial membrane potential ΔΨ has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells thus represents only the probe accumulated in mitochondria. Fibroblasts and cybrids were used as a model cell systems and optimal conditions for digitonin treatment and staining by TMRM were described. The TMRM signal collapsed by valinomycin, KCN and antimycin A and FCCP titration was used to gradually lower ΔΨ and characterise the stability of ΔΨ. The method is suitable for sensitive measurement of ΔΨ in different types of cultured cells.

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