Apoptosis and Mitochondrial Membrane Potential Assay

The apoptosis analysis was performed using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN BioTech, Nanjing, China) following the aforementioned method [28 (link)]. The excitation (Ex) and emission (Em) wavelengths are 488 nm and 530 nm, separately. The FITC positive/PI positive and FITC positive/PI negative cells were considered as apoptotic cells. The negative control was shown in Additional file 2: Fig. S1. The mitochondrial membrane potential (ΔΨm) change was also determined by flow cytometry. Briefly, cells were collected and incubated with 10 µg/mL JC-1 staining solution (Yeasen, Shanghai, China) for 15 min at a cell culture incubator. Then, cells were analyzed by flow cytometry immediately (Ex = 488 nm, Em = 530 nm). The rate of Q2 quadrant represented no loss of ΔΨm.