4.4.1. Cell Cycle Analysis

Cells in the different cell cycle stages have various amounts of DNA, i.e., the cells might possess 2 sets, 2 to 4 sets or 4 sets of chromosomes (2n, 2 to 4n, 4n) in the G1, S and G2/M phases, respectively. In this investigation, we permeabilised RMS cells using ethanol, stained chromosomes with propidium iodide (PI) and estimated the amount of DNA by measuring PI fluorescence intensities, since the number of chromosomes is proportional to the amount of PI. Cells were seeded in 6-well plates at 5 × 10⁵ cells/well and allowed to adhere overnight. Cells were treated with drugs for 24 h and then washed with PBS and trypsinised. The non-adherent cells (from the washing step) and adherent cells (from the trypsinisation step) are collected and centrifuged at 500× g for 5 min and washed with PBS (Splitting step). The cells were fixed in 500 µL of 70% ethanol for 30 min on ice and resuspended with 500 µL of PBS containing 40 µg/mL propidium iodide staining solution (PI), 0.1 mg/mL RNase A and 0.1% Triton X-100 followed by incubation at room temperature (RT) for 30 min. Finally, the cells were subjected to flow cytometry analysis using a BD FACSCalibur flow cytometer by evaluating cells using a 488 nm laser for excitation and a bandpass filter at 585/42 nm for emission (FL2-A channel).