Modfit lt dna analysis program

The cell cycle distribution was analyzed using flow cytometry with propidium iodide (PI) staining. Briefly, lentivirus-transduced cells (3×10⁵ cells/dish) were seeded in 6-cm dishes and incubated at 37°C for 40 h. Cells were harvested following trypsinization, washed with PBS and fixed in 70% cold ethanol overnight at 4°C. Cells were subsequently collected by centrifugation and resuspended in PBS containing 100 µg/ml of DNase-free RNase and 50 µg/ml PI (Sigma-Aldrich), and incubated in the dark at room temperature for 1 h. Finally, the suspension was subjected to FACSCalibur flow cytometer analysis (BD Biosciences, San Jose, CA, USA). The fractions of the cells in the G₀/G₁, S and G₂/M phases were analyzed with the ModFit LT DNA analysis program (Verity Software House, Topsham, ME, USA).

The cell-cycle distribution was analyzed by flow cytometry using propidium iodide (PI) staining. After lentivirus infection, U251 cells were seeded in a volume of 5 mL at a density of 2×10⁵ cells/well in 6 cm dishes. Cells were harvested after 40 hours of culture, and fixed in 70% ice-cold ethanol overnight at 4°C. After washing thrice with PBS, the cells were stained to determine DNA content using 300 μL PBS containing 50 μg/mL PI and 50 μg/mL pre-boiled ribonuclease A. The suspension was incubated in the dark at room temperature for 30 minutes and then subjected to flow cytometry using a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed with the ModFit LT DNA analysis program (Version 4 for Modfit LT, Verity Software House, Topsham, ME, USA).