Elvira Zakirova Each cell has different number of mitochondria which are of varying fitness dictated by many factors. The variation you see may be due to both factors. Use of a mito-specific dye such as mitotracker green to estimate quantity of mito per cell along with TMRM should considerably correct for variation you are seeing. I have done FRET using these two dyes and confocal microscopy and noticed in healthy cells many mitos do not have strong signals for mmp and some none, mito count was also variable. Hope this helps.

Agreed with Rabeah Al-Temaimi. Also, your cells could be stressed. It is better to start with a freshly thawed cell line.

Best wishes!