Cell Cycle Analysis of Riluzole-Treated Cells

Cell cycle analysis was performed as we described elsewhere^{40 (link)}. In brief, cells (5 × 10⁵) were seeded in cell culture dishes. After 24 h, the medium was removed and replaced with fresh medium containing riluzole 0–20 µM for 48 h. Cell cycle analysis was performed by measuring the amount of propidium iodide (PI) in ethanol fixed cells. In brief, cells were harvested by trypsinization and fixed with cold 70% ethanol for 24 h. Cells were rinsed 3 times with ice-cold PBS and resuspended in 1 ml of permeabilizing solution (Triton X 100 (0.25%), sodium azide (0.01%) and RNAs A (100 μg/μl Sigma-Aldrich) in PBS for 10 min. Cells were rinsed once with PBS, resuspended with 1 ml of PBS with PI (2.5 mg/ml) and incubated for 15 min at 4 °C. Cell cycle analysis was performed using a flow cytometer (Becton Dickinson).