Annexin 5 fluorescein isothiocyanate annexin fitc

Cell death was evaluated using Annexin V-fluorescein isothiocyanate (Annexin-FITC, BD Biosciences) and propidium iodide (PI, 2 μg/ ml, Sigma-Aldrich) followed by biparametric FACS analysis. Cells were stained with 5 μl of Annexin V-FITC and/or PI for 10 minutes at room temperature and washed once with binding buffer (10mM Hepes/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). The percentage of positive cells determined over 10,000 events was analyzed by a FACScan cytofluorimeter using the CellQuest software.

Apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate (annexin-FITC) and propidium iodide (PI) detection kit (BD Biosciences, San Jose, CA, United States). A549 and HepG2 cells were cultured in T25 flasks for 48 h. A549 cells were treated with 0.75 (Fa 95) of Cur, TQ, DIM, and their double (Cur + TQ, Cur + DIM, and TQ + DIM) and triple (Cur + TQ + DIM) combinations in 10 ml of medium for 24 h (Table 2). Also, HepG2 cells were treated with 0.75 (Fa 95) of Cur, TQ, and DIM and their double and triple combinations in 10 ml of medium for 24 h (Table 2). Cells were collected and centrifuged at 500 ×g for 5 min at room temperature after their trypsinization. The pellet was rinsed twice with phosphate-buffered saline (PBS) and then resuspended in a proper volume of binding buffer. After adding 10 μl of annexin V-FITC followed by gentle mixing, incubated for 15 min at room temperature in the dark and washed. The fluorescence intensity of FITC was carried on a FACSCalibur™ (Becton Dickinson) instrument using Cell Quest software (El-Far et al., 2020a (link); El-Far et al., 2021 (link)).