Cell Cycle Progression Analysis of Glutamine

The effect of glutamine on cell cycle progression was assessed by using Cellometer (Wang et al. 2014 (link); Nexcelom, Lawrence, MA, USA). Cells were plated at a density of 1.5×10⁵ cells/well in six-well plates overnight, and then treated with various concentrations of glutamine for 48 h. Cells were collected by 0.05% Trypsin (Gibco), washed with PBS solution, fixed in a 90% methanol solution and then stored at −20 °C until cell cycle analysis was performed. On the day of analysis, the cells were washed with PBS and centrifuged, resuspended in 50 μl RNase A solution (250 μg/ml) with 10 mM EDTA, followed by incubation for 30 min at 37 °C. After incubation, 50 μl of propidium iodide (PI) staining solution (2 mg/ml PI, 0.1 mg/ml azide, and 0.05% Triton X-100) was added to each tube and incubated for 10 min in the dark. The cell cycle was detected by Cellometer. The cell cycle progression was analyzed by the FCS4 Express Software (Molecular Devices, Sunnyvale, CA, USA).