Cell cycle progression analysis via quantification of PI staining

PI is a fluorescent dye used to quantify DNA content at 488 nm excitation.27 Flow cytometry employing PI was used to distinguish cells in the different cell cycle phases. After exposure, cells were trypsinized and washed in ice-cold PBS and resuspended in 200 µL ice-cold PBS. Cells were fixed in ice-cold 70% ethanol and incubated overnight at 4°C. Cells were resuspended in PBS containing 0.01% Triton X-100, PI (40 µg/mL) and RNase A (100 µg/mL) and incubated for 40 min at 37°C. PI fluorescence (detected at 590 nm) was measured using the fluorescence channel 3 (FL3) on the FC500 system flow cytometer (Beckman Coulter, Brea, CA, USA) after being excited at 488 nm. Cell cycle distributions were analyzed with Cyflogic flow cytometry analysis software (Beckman Coulter) using the DNA content per cell which illustrates the sub-G₁, G₁, S, G₂/M fractions.