Apoptosis Quantification by Flow Cytometry

For flow cytometry analysis, cells were seeded on 6-well plates at 5×10⁵ cells/well, with three replicate wells per experimental group. After a 48 h culture period at 37°C, the cells were harvested and washed twice with phosphate buffer, resuspended in 300 µl of binding buffer, and reacted with 5 µl of Annexin V-allophycocyanin (APC) and 5 µl of hypotonic propidium iodide (PI) solution for 10 min at room temperature. Then, 300 µl binding buffer solution was added and the cells were examined via flow cytometry for 10 min. The cells were analyzed using a BD flow cytometer (BD Via; BD Biosciences) (15 (link)). FlowJo software (v10.0; BD Biosciences) was used to analyze these data. The apoptosis rate was calculated as the percentage of early + late apoptotic cells. An Annexin V-APC/PI apoptosis kit (MultiSciences Biotech, Co., Ltd.) was used for the cell apoptosis assay.