MitoTracker Assay for Mitochondrial Membrane Potential

The assessment of ΔΨm was performed according to Ref. [95 (link)]. Briefly, cells were incubated for 20 min at RT in the dark, with a deep-red MitoTracker (20 nM final concentration) compound (cat #M22426, Thermo Scientific, Waltham, MA, USA). Cells were analyzed using fluorescence microscopy Floid Cells Imaging Station microscope (cat# 4471136, Life Technologies, Carlsbad, CA, USA), or a BD LSRFortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was conducted 3 times, and 10,000 events were acquired for analysis. MitoTracker highly positive cells were selected located between 10⁴ and 10⁶. No discrimination by complexity was made. Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software (BD Biosciences, Franklin Lakes, NJ, USA).