Annexin 5 fitc pi propidium iodide apoptosis detection kit

Cell death was assessed using the Annexin V-FITC/PI (propidium iodide) Apoptosis Detection Kit (KGA108; Keygen Biotech, Nanjing, China). HeLa cells were treated with control medium, dorsomorphin, 17-AAG, MG132, 17-AAG + dorsomorphin, or MG132 + dorsomorphin for 24 h. After staining with annexin V-FITC/PI, flow cytometric analysis was performed using a flow cytometer (Beckman Coulter Inc., Brea, CA, USA).

Apoptosis was quantified by measuring surface exposure of phosphatidylserine in apoptotic cells using an annexin V-FITC/PI (propidium iodide) apoptosis detection kit (KeyGEN Biotech, Nanjing, China) according to the manufacturer׳s protocol. Briefly, after A549 cells were treated with the indicated concentrations (40 and 80 μmol/L) of EFL2 for 48 h, the cells were collected and washed twice with ice-cold phosphate-buffered saline (PBS). Then 5×10⁵ cells were resuspended with 0.5 mL binding buffer containing Annexin-V (1:50 according to the manufacturer׳s instruction) and 40 ng/sample of PI for 30 min at 37 °C in the dark. Subsequently, the cells were assayed by flow cytometer (Becton Dickinson, USA) and analyzed with the CellQuest software (Becton Dickinson, USA). At least 10,000 cells were analyzed for each sample. The apoptosis rate was calculated using the following formula¹⁴ (link): Apoptosis rate (%)=(Number of apoptotic cells/Number of total cells observed)×100.