Fluorescence activated cell sorting flow cytometry

Cells were seeded into 6-well plates and treated with sorafenib for 24 h. 3 × 10⁵ treated cells were seeded into 6-well plates and cultured for 48 h at 37°C to assess the cell cycle and apoptosis. The cells for cell cycle analysis were digested using trypsin (Hyclone), washed twice with phosphate-buffered saline (PBS), and fixed in 70% ethanol overnight at 4°C. Then the cells were centrifuged at 500 × g for 5 min, washed twice with cold PBS, and centrifuged. Cell cycle analysis was performed through fluorescence-activated cell sorting flow cytometry (Beckman Coulter, Palo Alto, CA, USA) after treating the cells with RNase A (0.1 mg/mL) and propidium iodide (PI, 0.05 mg/mL) purchased from 4A Biotech (Beijing, China) for 30 min at 37°C. Following the instructions of the manufacturer, cells were harvested and were stained with Annexin V-FITC/PI (KeyGEN Biotech, Nanjing, China) for the analysis of apoptosis. Then the cells were acquired by flow cytometry (FACScan, BD Biosciences, USA) and analyzed by FlowJo 7.6.1.

SKOV3/PTX and HeyA-8/PTX cells were seeded into six-well plates and treated with PTX for 48 h. To assess the cell cycle and apoptosis, 3 × 10⁵ treated cells were seeded into six-well plates and cultured for 48 h at 37°C. The cells for cell cycle analysis were digested using trypsin (HyClone), washed twice with phosphate-buffered saline (PBS), and fixed in 70% ethanol overnight at 4°C. The cells were centrifuged at 500 × g for 5 min, washed twice with cold PBS, and centrifuged. After treating with RNase A (0.1 mg/mL) and propidium iodide (PI, 0.05 mg/mL) purchased from 4A Biotech (Beijing, China) for 30 min at 37°C, cell cycle analysis was performed through fluorescence-activated cell sorting flow cytometry (Beckman Coulter, Palo Alto, CA, USA). For the analysis of apoptosis, cells were trypsinized followed by two PBS washing steps. The cells were stained using the annexin V/PI detection kit (4A Biotech, Beijing, China) for 5 min at room temperature. The apoptotic cells were measured using flow cytometry (Beckman Coulter). All experiments were repeated at least three times.

SKOV3/PTX and HeyA-8/PTX cells were seeded into 6-well plates and treated with PTX for 48 h. To assess the cell cycle and apoptosis, we seeded 3 × 10⁵ treated cells into 6-well plates and cultured them for 48 h at 37°C. The cells for cell-cycle analysis were digested using trypsin (Hyclone), washed twice with phosphate-buffered saline (PBS), and fixed in 70% ethanol overnight at 4°C. The cells were centrifuged at 500 × g for 5 min, washed twice with cold PBS, and centrifuged. After treatment with RNase A (0.1 mg/mL) and propidium iodide (PI, 0.05 mg/mL) purchased from 4A Biotech (Beijing, China) for 30 min at 37°C, cell-cycle analysis was performed through fluorescence-activated cell sorting flow cytometry (Beckman Coulter, Palo Alto, CA, USA). For the analysis of apoptosis, cells were trypsinized followed by two PBS washing steps. The cells were stained using the Annexin V/PI detection kit (4A Biotech, Beijing, China) for 5 min at room temperature. The apoptotic cells were measured using flow cytometry (Beckman Coulter). All experiments were repeated at least three times.