L, [A+B], and D cybrids were plated in 24-well plates (100,000 cells/well), incubated 24 h and, treated with 0 or 40 µM of cisplatin for another 48 h. JC-1 reagent (5, 5′, 6, 6′-tetrachloro-1,1′, 3, 3′- tetraethylbenzimidazolylcarbocyanine iodide) (Biotium, Hayward, CA) was added to cultures for 15 min. Similar to the study conducted by Patel et al. a Gemini XPS Microplate Reader (Molecular Devices) was used to measure fluorescence for red (excitation 550 nm and emission 600 nm) and green (excitation 485 nm and emission 545 mm) wavelengths. Intact mitochondria with normal ΔΨm fluoresced red, while cells with decreased ΔΨm fluoresced green. Experiments were then analyzed in quadruplicate, and the entire experiment was repeated twice. All values were normalized to the average of the untreated-L cybrids, and cisplatin-treated cybrids were compared to untreated cybrids using a two-tailed t-test to assess for statistical significance (GraphPad Prism Software, Inc.) (Patel et al., 2019 (link)).
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