Jc 1 fluorescent probe

The 1 × 10⁶ cells were stained with 2 μM of JC-1 fluorescent probe (Biotium Inc., Hayward, CA, USA) for 30 min at 37 °C, centrifuged at 13,000× g for 5 min and resuspended in 0.5 mL PBS. Red fluorescence (λ excitation = 550 nm, λ emission = 600 nm), indicating polarized and undamaged mitochondria, and green fluorescence (λ excitation = 485 nm; λ emission = 535 nm), indicating depolarization and damaged mitochondria, were read using a Synergy HT Multi-Detection Microplate Reader, and expressed as relative fluorescence units (RFUs). Results were expressed as the percentage of green (depolarized)/red (polarized) mitochondria [32 (link)]. The opening of the mPTP, a second index of mitochondria depolarization and damage, was quantified in 1 × 10⁶ cells via flow cytometry (Guava EasyCyte equipped with the InCyte software v. 3.3, Millipore, Billerica, MA, USA) using the Mitochondrial Permeability Transition Pore Assay Kit (BioVision, Milpitas, CA, USA), as per manufacturer’s instructions. Autofluorescence of unstained cells was subtracted as blank. Results were expressed as the percentage of fluorescent cells over total cells.

Staining with JC-1 fluorescent probe (Biotium Inc., Freemont, CA, USA) was performed as detailed in Riganti et al. [19 (link)]. The resulting fluorescence units were used to calculate the percentage of green-fluorescent (i.e., depolarized) mitochondria versus red-fluorescent (i.e., polarized) mitochondria.