Annexin 5 apc 7 aad double staining

Cells were subjected to three different treatments (Lv-shTRIM24, Lv-shCon, and Con). After 72 h of incubation, cells were washed and reseeded in 6-cm dishes at a density of 80,000 cells/dish before detection. After 24 h, the cells were collected and subjected to Annexin V-APC/7-AAD double staining according to the manufacturer’s instructions (KeyGen Biotech, Nanjing, China).

Cells transfected with siMCM6 for 48 h were digested with trypsin without ethylene diamine tetra-acetic acid (EDTA) and the cell suspension was collected and washed with PBS twice. For cell apoptosis analysis, the cell suspension was subjected to Annexin V-APC/7-AAD double staining (KeyGen Biotech) and incubated for 15 min. Apoptotic cells were evaluated within 1 h by flow cytometry (FACScan; BD Biosciences) according to the manufacturer's instructions. For assessment of the results, mechanically damaged cells were in the upper left quadrant and negative normal cells were in the lower left quadrant; the early apoptotic cells were in the lower right quadrant and late apoptotic cells were in the uppper right quadrant. For cell cycle detection, the cells were fixed with 95% pre-cold ethanol for 2 h, and then washed with PBS twice. The cells were supplemented with 2 µl RNase A (2.5 mg/ml) and 15 µl propidium iodide (PI, 15X) (KeyGen Biotech), followed by detection with flow cytometry at 488 nm.