Annexin 5 fitc reagent

Viability, apoptosis, and necrosis were evaluated by Annexin V/propidium iodide (PI) staining. For this purpose, cells were seeded into 60-mm Petri dishes as 5 × 10⁵ cells per dish and incubated overnight to allow attachment. Cells were then treated with curcumin at respective IC₅₀ doses for 72 h. For Annexin V/PI staining, cells were detached by trypsinization, washed once with Dulbecco’s phosphate-buffered saline solution (DPBS, Thermo Fisher Scientific, #14190144), and suspended in 1X ice cold Annexin V Binding Buffer (BioVision Inc., #1006) followed by labelling with 1 μL Annexin V-FITC reagent (BioVision Inc., #1001) and 1 μL PI solution (Thermo Fisher Scientific, #P3566, diluted to 250 μg/mL in DPBS) by incubating for 10 min under dark conditions at room temperature. 2.5 × 10⁴ cells per test were read with Navios flow cytometry system (Beckman Coulter Inc.) and results were analyzed by Kaluza software (version 2.1). All experiments were performed as triplicates.