Annexin 5 fitc pi apoptosis kit

A propidium iodide (PI) cell cycle kit (Keygentec, China) was used to investigate the cell cycle, and an Annexin V-FITC/PI apoptosis kit (Keygentec, China) was used to evaluate the cell apoptosis. The treated or transfected HK-2 cells were cultured to a density of 80%-90% in 6-well plates, and collected after trypsin digestion. Then, these cultured cells were washed with PBS, and collected for detection. The apoptosis was detected by re-suspending the cells in 500 μL of binding buffer. Then, a mixture of 5 μL of PI and 5 μL of Annexin V-FITC was added into the binding buffer, and incubated for 30-60 min at room temperature in the dark. Afterwards, the apoptosis was examined using a flow cytometer. Apoptosis in the early stage was indicated by the PI-negative/Annexin V-positive staining, while more advanced apoptosis was indicated by the PI-positive/Annexin V-positive staining. Next, cell cycle assays were performed on cells washed with PBS, and these cells were collected and fixed in 1 mL of ice-cold 70% ethanol overnight at 4°C. Then, these cells were incubated with PI and RNase A (at a ratio of 9:1) at 37°C for 60 min. The cell experiments were repeated in triplicate. The apoptosis data obtained from the flow cytometry were analyzed using the FlowJo V10 software, while the cell cycle-related flow cytometry data were analyzed using the Modfit LT 5.0 software.