Annexin 5 fitc pi apoptosis detection assay kit

Annexin V FITC/ PI apoptosis detection assay kit (Invitrogen, USA) was used to determine the percentage of cells within a population that are actively undergoing apoptosis. In brief, cells (1 × 10⁶ cells/mL) were harvested after treatment with wtHALT-1 and camptothecin for 24 h using IC_50. Treatment with mHALT-1 was also carried out using the same test concentrations as wtHALT-1. Apoptosis was detected by initially staining the cells with 0.1% (v/v) Annexin V and 100 µg/mL of propidium iodide solution followed by flow cytometry analysis. Stained cell suspensions were incubated in the dark for 15 min at room temperature, 400 µL of 1×  binding buffer was added to each tube and analyzed using NovoCyte ™ 2060 flow cytometer (ACEA Biosciences, USA).

Annexin V and propidium iodide staining were conducted employing an annexin V FITC/PI apoptosis detection assay kit (Invitrogen, Carlsbad, CA, USA). Cells were initially plated in 6-well dishes at a density of 5 × 10⁵ cells/well. OPT-0139 was introduced at concentrations of 0, 1, and 10 µM, and the cells were subsequently incubated for an additional 48 h. Following trypsinization for cell detachment, the cells were harvested through centrifugation, washed with 1 mL of PBS, stained in accordance with the manufacturer’s instructions, and subjected to analysis using a flow cytometer (FACSCalibur and BD CellQuest Pro™ software version 5.0, BD Bioscience, San Jose, CA, USA).