Annexin 5 fitc pi kit

The proportion of AnV + cells (early apoptosis), AnV+/PI + cells (post-apoptotic necrosis), and PI + cells (necrosis) was determined using an Annexin V-FITC/PI kit (Beckman Coulter, Brea, CA, USA) and Epic XL flow cytofluorimeter (Beckman Coulter) in strict accordance with the standard procedure stated in the manufacturer's manual. At least 10,000 events were analyzed.

The apoptosis/necrosis assay was employed to identify the mechanism of cell death induced by P3C [20 (link)]. One hundred thousand cells (MDA-MB-231) per well in 1 mL of culture media were seeded in 24-well plates. Cells were incubated overnight at 37 °C in a 5% CO₂ atmosphere, and the following treatments were added on the next day: P3C CC₅₀ and 2× CC₅₀, 1% v/v DMSO (vehicle control), and 1 mM H₂O₂ (positive control for death). Cells were exposed to treatments for 24 h to subsequently use the Annexin V-FITC/PI kit following the manufacturer’s instructions (Beckman Coulter, Miami, FL, USA). Cells were harvested and placed in flow cytometer tubes and centrifuged for 5 min at 262× g. Supernatants were decanted, and 100 μL of a mixture of ice-cold 1× binding buffer containing PI and Annexin V-FITC was added to each tube by gentle resuspension. Tubes were incubated on ice in the dark for 15 min, followed by the addition of 300 μL of ice-cold 1× binding buffer. Cells were immediately analyzed by flow cytometry (Gallios, Beckman Coulter, Brea, CA, USA), collecting around 10,000 events/cells per sample. The data collection and analysis were accomplished using the Kaluza 1.3 software (Beckman Coulter).