Cell cycle distributions were determined by flow cytometry through staining of the cells with propidium iodide (PI). Briefly, Hep3B cells were harvested at 48 h or 72 h after siRNA treatment, then washed with 1× PBS and resuspended in 75% cold ethanol at 4 °C overnight. After a further wash with 1× PBS, fixed cells were treated with RNase for 30 min, and incubated with 30 μg/mL PI for one hour in the dark. The cells were analyzed with a Beckman Coulter Cytomics FC500MPC Flow Cytometry Analyzer.
The percentage of apoptotic cells was assessed with an eBioscience™ Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions (Invitrogen, 88-8005-72). Hep3B cells transfected with siRNA or sgRNA constructs were harvested and washed with cold PBS, then resuspended in 100 μL binding buffer containing 5 μL Annexin V-FITC. After 15 min incubation in the dark, cells were centrifuged at 1000 rpm for 5 min and resuspended in 500 μL binding buffer containing 5 µL of PI for 15 min in the dark at RT. The apoptosis was analyzed by a Beckman Coulter Cytomics FC500MPC Flow Cytometry Analyzer.
The percentage of apoptotic cells was assessed with an eBioscience™ Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions (Invitrogen, 88-8005-72). Hep3B cells transfected with siRNA or sgRNA constructs were harvested and washed with cold PBS, then resuspended in 100 μL binding buffer containing 5 μL Annexin V-FITC. After 15 min incubation in the dark, cells were centrifuged at 1000 rpm for 5 min and resuspended in 500 μL binding buffer containing 5 µL of PI for 15 min in the dark at RT. The apoptosis was analyzed by a Beckman Coulter Cytomics FC500MPC Flow Cytometry Analyzer.