Facs calibar flow cytometry

After cells were cultured in SC-G at the special time, harvested and stained by JC-1 (dissolved in DMSO, final concentration 1 μg/mL, Sigma, United States) at 37°C for 30 min (Ratnakumar and Tunnacliffe, 2006 (link); Pina-Vaz and Rodrigues, 2010 (link); Dong et al., 2015 (link); Liang et al., 2018 (link); Lee et al., 2019 (link)). Samples were measured by flow cytometry (FACS Calibar flow cytometry, BD, United States). Gated region R1 (Ex = 488 nm, Em = 525 ± 20 nm) includes cells with intact mitochondrial membranes and gated region R2 (Ex = 514 nm, Em = 585 ± 20 nm) depicts cells with decreased of mitochondrial membrane potential. All samples were obtained from three independent experiments.

To measure the mitochondrial membrane potential (MMP), cells were cultured in YPD medium (containing 5 mm/L H₂O₂) for 1 h. Then cells were incubated with a cationic lipid fluorescent dye (JC-1, dissolved in DMSO, final concentration: 1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) at 30 °C for 30 min, and the cells were measured by flow cytometry (FACS Calibar flow cytometry, BD, San Jose, CA, USA).