Cxp system software

Cells were seeded, treated, and incubated for the indicated periods. The cell monolayer was harvested, washed in PBS, and fixed for 30 min in 1 mL of ice-cold 70% ethanol. The fixed cells were then pelleted, rinsed with PBS, incubated in the presence of RNase A for 15 min at 37 °C, stained with propidium iodide, and kept in the dark. The stained cells were analyzed for DNA content using a Cytomic FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Approximately 20,000 cells per sample were analyzed using excitation at 488 nm and emission at 617 nm. The percentage of cells in each phase of the cell cycle was determined using the CXP System Software (Beckman Coulter, Miami Lakes, FL, USA).

PANC-1, MIA-PaCa2 and SW1990 cell lines were treated with rIL-37 (100 ng/ml), Gem (2 μM) and Gem plus rIL-37 for 24 h and divided into four groups. Prior to the treatment, cells were cultured overnight in serum-free conditions to synchronize cell growth. Cells were trypsinized and fixed in cold 70% ethanol for 10 min and then stained with propidium iodide (PI) solution at room temperature for 15 min. Approximately 10,000 sample cells were analyzed using flow cytometer (Beckman Coulter) with excitation at 488 nm and emission at 617 nm. The percentage of cells in each phase of the cell cycle was determined using CXP System Software (Beckman Coulter).
PANC-1, MIA-PaCa2 and SW1990 cell lines treated with rIL-37 or/and Gem were analyzed for phosphatidylserine exposure by an annexin-V FITC/PI double-staining method using a commercial kit (BD Biosciences, Cat. 556570) according to the manufacturer's instructions. A minimum of 5,000 cells were then analyzed by FACScan with Cell Quest software (Beckton Dickinson) for acquisition and analysis.