Annexin 5 apc apoptosis detection kit

Cells were plated in 6-well plate with 1*10⁵ cells/well. After 12 hours, cells were infected with lentivirus vectors. After 48 hours incubation, cells were harvested, washed with PBS and incubated with Annexin V and PI, using the Annexin V-APC apoptosis detection kit (KGA1022, KeyGen, China). The flow cytometry analyses were performed with CytoFLEX instrument (Beckman Coulter, USA).

All group cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. For cell cycle detection, cells were digested with trypsin without Ethylene Diamine Tetraacetic Acid (EDTA). After fixing with 1ml 70% ethanol overnight, cells were centrifugated and the cell pellets were stained with 2 μl 50ug/ml PI and 1 μl 100μg/mL RNase A for 30 min at 4°C in darkness. Subsequently, cells were analyzed by Flow cytometry. For cell apoptosis assay, the digested cells were stained with Annexin V-APC Apoptosis Detection Kit according to the manufacturer’s protocols (KeyGEN, Nanjing, Jiangsu, China). Then the apoptosis rates were detected using a flow cytometer.

For analysis of cell cycle, cells transfected with plasmids and specific siRNAs were cultured in 6‐well plates until they reached 80% confluency. Cells were then digested with trypsin without ethylene diamine tetraacetic acid and washed twice with phosphate buffered saline (PBS). After fixing with 75% ethanol at −20°C for 24 hours, the cell precipitate was stained with PI in PBS for 30 minutes in darkness. Next, all cells were analysed with a flow cytometer (Millipore, USA). For apoptosis detection, cells of all groups were seeded in 12‐well plates. After fusion growth, the cells were transferred to a centrifuge tube and washed twice with PBS. Apoptotic cells were detected with the Annexin V‐APC Apoptosis Detection Kit according to the manufacturer's instructions (KeyGEN, Nanjing, Jiangsu, China). Subsequently, the apoptosis rates were measured using a flow cytometer.

Single‐cell suspensions were generated following trypsinization and resuspension. Before cell cycle experiments, CRC cells were fixed in 70% ice‐cold ethanol for at least 2 hours and then stained with propidium iodide according to the manufacturer's protocol. For apoptosis analysis, cells were stained with Annexin V‐APC and propidium iodide using Annexin V‐APC Apoptosis Detection Kit (KeyGEN). All samples and statistics were analyzed using a FACS caliber flow cytometer (BD Biosciences), BD Cell Quest software, and LSRFortessa (BD Biosciences).