V fitc apoptosis detection kit

Apoptosis was detected after 48 h of cell culture using V-FITC apoptosis detection kit (BD Pharmingen TM, Franklin Lakes, NJ, USA). MDA-MB-231 cells were treated with the predetermined IC₅₀s of the most potent compounds for 24 h, and the untreated group was included as a control group. After treatment, cells were collected, washed with ice-cold PBS and centrifuged at 200× g for 5 min. The cell pellet was suspended in 200 μL of annexin V-FITC/PI solution for 15 min in the dark. The stained and Annexine V propidium iodide negative cells were evaluated via CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, USA). Ten thousand cells (gated events) were captured for each sample. The mean fluorescence intensity was analyzed with Cytoexpert (Beckman Coulter).

The PIG3V cells were inoculated in a 6-well plate (3 × 10⁶ cells per well) for 24 to 48 hours. After each time point, the supernatant was discarded, and 1.5 ml 254 medium was added to each well. Cells were then incubated with 0, 0.4, 0.8, 1.6, and 3.2 mM H₂O₂ (Sigma, China) to induce apoptosis. After 24 hours of treatment, according to the manufacturer's protocol, the V-FITC apoptosis detection kit (BD, USA) was used to distinguish between apoptotic and necrotic cells. The apoptosis rate was further measured by flow cytometry (Beckman Coulter, USA).