Cells were harvested and washed with phosphate-buffered saline (PBS) for twice. For cell apoptosis, Annexin V-PE and 7-aminoactinomycin D (7-AAD) solution (Keygentec, Nanjing, China) were added to each sample for incubating 15 min in the dark. For cell cycle analysis, cells were mixed with 70% ethanol for overnight at 4 °C, washed with cold PBS and centrifugally harvested at 1000 rpm for 5 min. The supernatants were resuspended in Propidium Iodide (PI) and Ribonuclease A (RNase A) staining buffers (Keygentec, Nanjing, China) for 15-20 min in the dark. The fluorescent signals were detected by flow cytometry (BD FACSCalibur) within 1 h. Living cells were in PE-/7-AAD- quadrant, early apoptotic cells were in PE+/7-AAD- quadrant and late apoptotic cells were in PE+/7-AAD+ quadrant.

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