Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis detection kit

Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit
(Vazyme, China) was adopted to evaluate the apoptosis of HCCLM3 and BEL7402 cells. The
cells were harvested 48 hours after transfection and the cell concentration was adjusted
to 1×10⁶ cells/ml with binding buffer. Subsequently, the cells were incubated
overnight with pre-cooled 70% ethanol at 4˚C. After being centrifuged, the cells was
re-suspended in binding buffer (200 μl). The re-suspended cells were then stained with PI
staining solution (5 μl) and Annexin V-FITC staining solution (10 μl) in dark for 15
minutes at room temperature. Afterwards, a MoFlo XDP flow cytometer (Beckman Coulter, USA)
was utilized to analyze apoptotic cells. Data were processed by BD FACSDiva™ software (BD
Bioscience, USA).

Cell apoptosis levels was detected using a Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Vazyme Biotech Co., Ltd.). In brief, the cells were incubated at a density of 3.5x10⁵ cells/well in 6-well plates and were collected and stained with 5 µl Annexin V-FITC and 5 µl PI solution in the dark for 15 min at room temperature after oxygen-glucose deprivation/reperfusion. Subsequently, the stained cells were analyzed using a flow cytometer (FACScan™; BD Biosciences) and Modfit software 3.2 (Verity Software House, Inc.).