The cells were digested with trypsin without EDTA. We used serum-containing medium to stop the digestion. After centrifugation at 1,000 rpm for 3 min, the supernatant was discarded and gently washed with cold PBS. Using preconstituted 1× binding buffer, we made a cell suspension with a final concentration of 1 × 107 cells/mL, added 10 μL of annexin V-PE (phycoerythrin), and cultured it for 15 min on ice in a dark room. Then, we added 10 μL of 7-aminoactinomycin D (7-AAD) with 380 μL of cold binding buffer. Cell apoptosis was detected within 1 h using flow cytometry.
