Apoptosis Detection in HepG2 Cells

Cell apoptosis was determined by the Annexin V/PI apoptosis detection kit (Solarbio). Annexin V can bind to phosphatidylserine on the surfaces of HepG2 apoptotic cells and is detected with a flow cytometer in the FITC channel, as a marker for apoptosis. PI can enter the dying or dead cells and is detected with a flow cytometer in PE channel, as a marker for cell death. The cells were segregated into four quadrants: Annexin V−/PI− (viable cells), Annexin V+/PI− (early apoptotic cells), Annexin V−/PI+ (necrotic cells), and Annexin V+/PI+ (late apoptotic cells). HepG2 cells were digested with trypsinization (Procell), centrifuged at 1000× g rpm for 5 min, and then collected. Subsequently, the cells were washed and resuspended in pre-cooled PBS and centrifuged for recollection. This elution step was repeated twice. Then, the cells were resuspended in 1 × binding buffer and incubated with Annexin V and PI staining solution at room temperature for 5 min. Cellular apoptosis was assessed via flow cytometry (Mindray, Shenzhen, China). Analysis was performed using Flow Jo software version 9.3 (Tree Star, Ashland, OR, USA) [38 (link),39 (link),40 (link)].