For annexin V-FITC/PI double staining, the cells were harvested and washed twice with cold PBS, and then the cells were stained with annexin V in binding buffer for 15 min at room temperature, followed by staining with PI for 5 min without annexin V. The cells were then analyzed by flow cytometry using the BD FACSCanto II Flow Cytometer (FACSCanto; Becton Dickinson). Data were acquired and analyzed by using the CELLQuest software (Becton Dickinson).

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