Annexin 5 apoptosis detection kit for flow cytometry

Aqueous, 50% and 100% ethanol Tritticum spelta and Salix mucronata plant extracts were tested for their pro-apoptotic activity. The annexin V apoptosis detection kit for flow cytometry (Sigma-Aldrich, MO, USA) was used. The annexin V assay was carried out in conjunction with propidium iodide (PI) staining. HepG2 and Caco-2 cancer cell lines were cultured for 24 h in a 25 cm² culture flask (1 × 10⁶ cells/well) with 66.6 μg/mL (the lowest IC₅₀) of the investigated extracts. After 72 h, cells were harvested by trypsinization and centrifuged at 1000 rpm for 5 min and then re-suspended in 1 × binding buffer prior to staining with 5 μL of annexin V and 10 μL of propidium iodide solution for 15 min at room temperature. The apoptosis-dependent anti-proliferative effect was determined by quantification of annexin-stained apoptotic cells using the FITC signal detector (FL1) against the phycoerythrin emission signal detector (FL2)^{20 (link)}.

An annexin V apoptosis detection kit for flow cytometry (Sigma-Aldrich, MO, USA) was used. The annexin V assay was carried out in conjunction with PI staining. HepG2 cells were cultured for 24 h in a 25 cm² culture flask (1 × 10⁶ cells/well) in the presence of different concentrations of the extract (IC₂₅, IC₅₀ and IC₇₅). After 24, 48 and 72 h, cells were harvested by trypsinization and centrifugation at 1000 rpm for 5 min and then re-suspended in 1x binding buffer prior to staining with 5 μl of annexin V and 10 μl of propidium iodide solution for 10 min at room temperature. FACS analysis was then carried out according to the manufacturer’s instructions, using a BD LSRFortessa™ Cell Analyzer (Becton Dickinson, NJ, USA). About 10,000 counts were recorded in each analysis [28 (link)].