Apoptosis assessment by flow cytometry, caspase assay, and Western blot

A flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) was used to measure apoptosis in terms of annexin V (Strong Biotect Corporation, Taipei, Taiwan)/7AAD [27 (link)] and a pancaspase activity assay kit (Abcam, Cambridge, UK) [23 (link)] assays. Apoptosis was also detected by Western blotting applying primary antibodies for the cleaved forms of poly (ADP-ribose) polymerase (c-PARP) and caspase 3 (c-Cas 3) (Cell Signalling Technology Inc., Danvers, MA, USA) (diluted 1:1000) and internal control mAb-β actin (Sigma-Aldrich, St. Louis, MO, USA) as described previously [14 (link)]. Cas 3/7 activity was determined by a Caspase-Glo^® 3/7 Assay (Promega; Madison, WI, USA) based on luminescent detection as described previously [28 (link)].