Cell experiment:

Cultures are exposed to Millipore-filtered solutions (0.22 μm) containing TMRM Perchlorate for 1 hr at 37℃ (except the experiment involving different durations of exposure to TMRM Perchlorate). After treatment, solutions are removed and growth media reapplied under sterile conditions, and cultures are post-incubated for 18 hours at 37℃ (except for the experiment involving analysis at different time points after exposure). Cells are then stained with 2 mg/mL bisbenzimide for 20 min at room temperature. Coverslips are subsequently washed in saline and imaged using 2P microscopy. Apoptotic cells are identified as brightly fluorescent nuclei under UV excitation indicating DNA fragmentation. Cell survivability is calculated as the percentage of live, unstained cells (±SD) in five microscopic fields per treatment[1].

Tetramethylrhodamine methyl ester (TMRM) (perchlorate) is a non-cytotoxic cell-permeant fluorogenic dye most commonly used to assess mitochondrial function using live cell fluorescence microscopy and flow cytometry.1,2,3 It has two excitation peaks at 515 and 555 nm and an emission peak in the red-orange range (575 nm). Due to the polarization of the mitochondrial membrane, TMRM is taken up into healthy mitochondria. However, when the membrane is depolarized, as in apoptosis, it is not taken up or is released from the mitochondria. Thus, the strength of the fluorescence signal in mitochondria is used to assess cell viability.

Reference:

1. Farkas, D.L., Wei, M.-d., Febbroriello, P., et al. Simultaneous imaging of cell and mitochondrial membrane potentials Biophys J. 56(6), 1053-1069 (1989).
2. Gandra, P.G., Nogueira, L., and Hogan, M.C. Mitochondrial activation at the onset of contractions in isolated myofibres during successive contractile periods J. Physiol. 590(15), 3597-3609 (2012).
3. Michea, L., Combs, C., Andrews, P., et al. Mitochondrial dysfunction is an early event in high-NaCl-induced apoptosis of mIMCD3 cells Am. J. Physiol. Renal Physiol. 282(6), F981-F990 (2002).