Annexin V-APC Apoptosis Assay

The Annexin V-APC Apoptosis Detection Kit (KGA1030; KeyGEN Biotech) was used to identify apoptotic cells as described in our previous studies and others [13 (link), 48 (link)]. In brief, the cells were seeded in 6-well plates (6×10⁵ cells/well). After incubation with MG for 24 h, cells were collected and washed twice with PBS. The cell suspensions were stained with annexin V and PI from the kit for 30 min at 4°C in the dark. The apoptotic cells were then quantified by flow cytometry analyzer (BD Biosciences, San Jose, CA). BD Accuri C6 software was used to analyze the data. Cell apoptosis was determined as either annexin positive or both annexin and PI positive by flow cytometry and the percentage of apoptotic cells was calculated.