Flow cytometry

One hundred thousand BPH-1 and PWR-1E cells were seeded into each well of a six-well plate, and rMIF (300-69, PeproTech, USA; 100 ng/ml), ISO-1 (S7732, Selleck, USA; 10 µM), celecoxib (S1261, Selleck, USA; 5 µM) or nothing was added into each well. The cells were collected on the second day. Then, the cells were added to 5 ml of 70% (precooled) ethanol and stored at 4°C to fix overnight. The fixed cells were collected, and RNAse (10 mg/ml in 1 mol/l Trisc1, pH 7.4) was added. The cells were then incubated for 30 min at 37°C in water. Propidium Iodide (PI) (450 μl PI per sample undiluted) was added to the cells and then incubated for 30 min on ice. Then, flow cytometry was used to analyze the cells, Cellquest software was used to obtain data, and Modfit software was used to analyze the cell cycle.