Cell experiment:

The entire experiment should be performed at room temperature because temperature will directly impact mitochondrial transmembrane potential and TMRE staining. Cells should never be placed, centrifuged, incubated, or washed at 4℃ or have ice-cold buffers or media added. Treat the cells with a cytotoxic stimulus. Harvest cells and resuspend at 5×105 cells/mL in culture medium containing 150 nM TMRE. Incubate for 5 min at room temperature in the dark. Add Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (5 μM final concentration) to an aliquot of untreated cells and incubate for 5 min at room temperature in the dark. Turn on the appropriate laser on the flow cytometer. Set up a histogram plot to detect TMRE using log scale[1].

TMRE is a mitochondria specific dye (λex=550 nm, λem=575 nm).

TMRE is a mitochondria specific dye (λex=550 nm, λem=575 nm)[1]. Multidirectional dynamic movement of TMRE is observed in epithelial cells and bidirectional dynamic movement is seen in the superficial cortical fiber cells of live bovine lenses. In the epithelium, the movement of TMRE fluorescence is up to 5 μm/min whereas in the superficial cortex the observed movement is up to 18.5 μm/min. The movement of TMRE fluorescence is abolished with treatment of the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP)[2].

[1]. Crowley LC, et al. Measuring Mitochondrial Transmembrane Potential by TMRE Staining. Cold Spring Harb Protoc. 2016 Dec 1;2016(12):pdb.prot087361. [2]. Bantseev V, et al. Confocal laser scanning microscopy imaging of dynamic TMRE movement in the mitochondria of epithelial and superficial cortical fiber cells of bovine lenses. Mol Vis. 2005 Jul 14;11:518-23.