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Annexin V/PI apoptosis detection

CU Caroline E. Ukwade
OE Osaretin A. T. Ebuehi
RA Rahmat A. Adisa
SS Santosh K. Singh
RS Rajesh Singh
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Apoptosis assay detection kit with propidium iodide (PI) (Biolegend, San Diego, CA) was used to investigate if plant extract could induce cell apoptosis in OvCa cells. Both (OVCAR-3 and SW 626) cells were seeded (1 × 106) in a six- well plate for 24 h and treated with an IC50 value of plant extract for 48 h. Further, cells were washed, harvested with 0.25% trypsin, and counted using the hemocytometer (Countess II FL, Life Technology, CA, USA). Subsequently, cells were washed twice with an ice -cold Annexin V binding buffer, and an equal number of cells (1 × 105) were stained with Annexin V (conjugated with FITC) (5 μL) and PI (10 μL) in dark for 15 min at room temperature, according to manufacturer instructions. The Guava Flow cytometer (EMD Millipore, Brillarica, MA, USA) was used to acquire (50, 000 events/ sample) the frequency of apoptotic cells and analyzed using FlowJo 10.0.06 software.

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