Annexin 5 fitc pi staining kit

Cell apoptosis was analysed using the Annexin V‐FITC/PI staining kit (Roche Applied Science). The treated cells were collected and labelled for 15 minutes with annexin V and propidium iodide (PI). The apoptotic cells were detected in a flow cytometer (Beckman Coulter).

BC cells were cultured (4 × 10⁴ cells/well) in 24-well plates, and 24 h after seeding cells were transfected with the constructs. 36 h after transfection, the cells were collected and stained using the Annexin-V-FITC/PI Staining Kit (Roche) and incubated for 15 min at room temperature in the dark. The cells were then evaluated by obtaining the PI- and Annexin V positive cells using a FACSCalibur Flow Cytometry System (BD Bioscience).

Melanoma cells were treated with frugoside at the doses or times indicated in the figures. Similarly, for the inhibitor experiments, melanoma cells were cultured in the presence or absence of NAC, DPI, or SB202190 to confirm the effect of frugoside. Cell death was measured by a FACS analysis after staining using the Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining kit (Roche, Nutley, NJ, USA). Cell viability was determined using a CCK-8 assay kit (Dojindo, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, melanoma cells were inoculated into a 96-well microplate, and a CCK-8 solution (10 μL/100 μL medium) was added to each well. After incubation for 1–4 h in a CO₂ incubator at 37 °C, the absorbance of each well was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) with a reference wavelength of 650 nm. Cell cycle distribution was determined by DNA staining with PI (Sigma). Cells were harvested and fixed in 70% ethanol. Cell pellets were suspended in PI and simultaneously treated with RNase at 37 °C for 30 min. The percentage of cells in different cell cycle phases was measured using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).