Z vad fkm

Transfected HeLa cells were harvested, counted, washed twice in PBS and 1 × 10⁶ cells were stained for 30 min with 1 μl 780eFluor of Fixable Viability Dye (eBioscience) in 1 ml PBS. For Z-VAD-FKM experiments, culture media was removed 6 h after transfection and replaced with 20 μM Z-VAD-FKM (Sigma) containing complete media. After PBS washing, cells were stained with 10 μl Annexin V-V450 (BD Biosciences, 560506) in 200 μl 1× binding buffer for 15 min. After washing, cells were fixed in 2% ice-cold paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized in 90% ice-cold methanol (Sigma-Aldrich) for 30 min. V5 staining was then performed using mouse anti-V5 Alexa-Fluor 647 conjugated antibody (clone SV5-Pk1, Biorad) and stained samples were acquired on a MACSQuant Analyzer (Miltenyi Biotec). Data were analyzed with FlowJo software (Tree Star Inc). Treatment with 100 μM etoposide (Sigma) in dimethylsulfoxide for 12 h was used as positive control.