Fcs software

A2780/CP70 and OVCAR-3 cells (1 × 10⁶/dish) were treated with galangin at different concentrations (10–40 μM) for 24 h, digested by trypsin and collected through centrifugation. Subsequently, cells were washed with cold PBS, suspended in 70% ethanol and stored at −20 °C. After being collected through centrifugation for 6 min at 1000 rpm, the cell pellets were washed with PBS again, and the PBS was removed. Then, they were incubated with 180 μg/mL RNase A at 37 °C for 15 min. Next, 50 μg/mL propidium (final concentration) was added for 15 min staining and then detection by flow cytometry (FACSCalibur system, BD Biosciences, Franklin Lakes, NJ, USA). Data were plotted and analyzed with FCS Software (De Novo Software, Los Angeles, CA, USA).

According to the method described in the manufacturer’s instructions of Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit. In brief, both OVCAR-3 and A2780/CP70 cells (10⁶/dish) treated with TS or RPMI-1640 (FBS free) for 24 h were washed with cold PBS twice and digested by trypsin. Cells were collected by 1000 rpm centrifugation for 8 min at 4 °C. Then the cell pellets were re-suspended in 1 mL 1× Annexin V binding buffer. An aliquot of 100 µL of the cell solution (around 2.0 × 10⁵ cells) was transferred to a 1.5 mL tissue culture tube. Subsequently, 5 µL of Annexin V-FITC was added into tubes and gently vortexed and incubated in the dark at room temperature for 15 min. Then 10 µL PI (1 mg/mL in PBS) was added to each tube and stained for 10 min when kept on ice. Following this, 290 µL of binding buffer was added to each tube and gently vortexed before detection. The samples were analyzed by flow cytometry (FACSCalibur system, BD Biosciences, San Jose, CA, USA). Data was plotted and analyzed using FCS software (De Novo Software, Los Angeles, CA, USA).

The test method of Cell cycle evaluation was according to our previous procedure [17 (link)]. In brief, after exposure with GA (control, 5, 10, 20 μM) for 48 h, the cells were trypsin-digested, centrifuged at 3000× g rpm for 10 min, PBS-washed, suspended in 70% ethanol, and stored at −20 °C. For cell cycle evaluation, the stored cell suspension was centrifuged at 1000× g rpm for 6 min before re-suspension in PBS, and centrifuged again to collect pellet, and finally exposed to 180 μg/mL RNase A at 37 °C for 15 min and 50 μg/mL propidium iodide stain for 15-min before flow cytometry (FACSCalibur system, BD Biosciences) evaluation. Data analysis was done with FCS software (De Novo Software, Los Angeles, CA, USA).