Annexin 5 and 7 aad apoptosis detection kit

GC cells were harvested at 48 h after transfection by trypsin and washed three times with cold phosphate-buffered saline (PBS). For the cell cycle assay, the cells were fixed in cold 70% alcohol overnight at 4 °C, washed and then stained with propidium iodide (PI) (Solarbio, Beijing, China) containing RNase A. After incubation in the dark at 4 °C for 30 min, the cells were analysed within 1 h. The percentages of various cell cycle phases (G0/G1, S and G2/M phases) were counted and compared. For the apoptosis assay, the cells were mixed with a double staining Annexin V and 7-AAD Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA) in the dark at room temperature for 15 min. Both experiments were analysed by flow cytometry analysis (BD) according to the manufacturer’s protocol.

Viable target and effector cells (CD56+, CD8+ and CD4+) were counted and resuspended in lymphocyte culture media (10% FBS, 1 mM sodium pyruvate, 0.5 mM 2-Mercaptoethanol, 1 mM HEPES, 100 u/ml penicillin, 100 u/ml streptomycin, 0.292 mg/ml L-Glutamine in RPMI). Target cells were cultured in four separate conditions: with media alone, with CD56+ cells, with CD8+ cells, and with CD4+ cells. Cells were cultured in 96-well plates at a ratio of 10 effector cells (50,000 cells) to 1 target cell (5,000 cells). Plates were briefly centrifuged and then cultured at 37°C and 5% CO₂ for 4 hours. Cells were then collected, washed, and resuspended in 100 µl of flow cytometry staining buffer. Monoclonal antibodies against CD45 were added and cells were incubated in the dark, with shaking, at room temperature for 25 minutes. Cells were washed with 2 mL of staining buffer. Cells were then stained with the Annexin V and 7-AAD Apoptosis Detection Kit (BD Biosciences, San Jose, CA) according to manufacturer’s instructions. Cells were analyzed immediately by flow cytometry as described above. The percent of cytotoxic cells was determined by the following equation: ((Target alone) – (Target + Effector))/Target alone)*100.