Flow cytometry: Cell cycle progression

Cell cycles G1, S, G2 were analysed in HT 29 cells using propidium iodide to stain the nucleus in order to determine the amount of DNA. Cells were seeded in 25cm² flasks and incubated at 37 °C for 24 h after which the cells were treated with 100, 500 and 1000 μg/mL extracts for 24 h, 48 h and 72 h. Cells were harvested and pelleted by centrifugation at 3500 rpm/6 min. The supernatant poured off and the 3 mL of ice cold 95% ethanol was added to each tube in a drop wise manner whilst vortexing each tube till the cell-ethanol mixture was homogenous. The specimens were stored at − 20 °C overnight. The ethanol was removed by centrifugation at 3500 rpm/6 min and the cells washed with 1 mL PBS at 6 min intervals. The sediment was re-suspended in 1 mL hypotonic DNA staining buffer, Propidium Iodide (PI) / RNase [Roche], and stored at 4 °C for 30 min before reading the specimens using a Becton Dickinson FACS Calibre benchtop flow cytometer with a 488 nm Coherent laser. For each sample at least 10,000 events were collected and aggregated cells were gated out. The number of gated cells in the G_1, G₂/M and S-phase is represented as %. (RNase solution: 250 mL distilled water, 0.25 g Tri NaCitrate, 750 μL Triton X-100, 0.025 g PI and 0.005 g RNase A).