Mitochondrial Dysfunction Assay via TMRE Staining

As done previously [36 (link), 37 (link)], cell media was collected 24 h after transfection and cells were washed with PBS then harvested with 0.25% Trypsin EDTA (ThermoFisher Scientific). Cells were then pelleted by centrifugation at 1000 RPM for 5 min, after which media was aspirated and cells were re-suspended in 300 μl of 100 nM tetramethylrhodamine ethyl ester (TMRE, Invitrogen) in 1x Annexin-V buffer (Invitrogen). Cells were incubated at 37 degrees C for 30–45 min, then analyzed on the FACSCanto-II (BD-Biosciences, University of Utah Flow Cytometry Core), where they were gated for proper morphology, GFP (excitation 488 nm, emission filter 530/35), and TMRE (excitation 561, emission filter 585/15) with FACSDiva software.