Fc500 mcl machine

RAW 264.7 cells were seeded and treated with designed doses of cypermethrin for 48 h. After treatment, cells were trypsinized, centrifuged at 300× g for 5 min and washed with phosphate belanced solution (PBS), then re-suspended in 1 mL of cold 70% (v/v) ethanol, and stored at 4 °C for at least 2 h. Then, the fixed cells were washed with 2 mL PBS and incubated for 15 min. After centrifugal, the cells were added 1 mL Regent A of cell cycle staining kit. Following mixing, cells were incubated in dark condition for 30 min at 37 °C. The cells were then analyzed using a FC500 MCL machine (Beckman Coulter, Brea, CA, USA).

For flow cytometry measurements of the cell cycle, 36 h-post transfection cells were trypsinized, centrifuged at 300 X g for 5 min, and fixed overnight in 70% cold ethanol at -20°C. After washing twice with PBS, the cells were resuspended in 500 μL of fresh PBS containing 50 μL of 2 mg/mL RNaseA and 10 μL of 1 mg/mL PI (Sigma). Cells were incubated for 15 min at 37°C. The cells were then analyzed immediately using a FC500 MCL machine (Beckman Coulter) at10,000 events/sample.

The Annexin V-FITC/PI kit (Multiscience) was used to analyze the extent of apoptosis. Briefly, cells were collected by trypsinization, washed three times with PBS, and then resuspended in 500 μL binding buffer with 5 mL Annexin V-FITC and 10 μL PI. Cells were incubated for 5 min in the dark at room temperature. The cells were then analyzed using a FC500 MCL machine (Beckman Coulter) at 10,000 events/sample.