2.6 Detection of apoptosis via annexin V–FITC/PI method
A549 cells in the logarithmic growth phase and in good growth condition were digested by trypsin, and the reaction was terminated by adding RPMI1640 fresh medium containing serum and centrifuged to make cell suspension. Inoculated in 6-well plates at 1×106cell/mL, 2 ml per well, and incubated at 37 ℃ for 24 h in a 5% CO2 incubator; When the cells grew to about 80%, they were treated with 300, 150, and 75 μg/mL WT andAaFla1 OE23 mutant crude extracts for 48 h. The cells were then collected, washed 3 times with 1xPBS, processed by centrifugation, and stained according to Annexin V-FITC/PI kit before being detected by flow cytometry for apoptosis.