Analyzing Cell Cycle in Neuro2A Cells

Neuro2A cells were seeded in six wells (5 × 10⁵ cells/well), 24 h later, plasmids of miR-30c-OE, miR-30c-KD, and vehicle control were transfected into Neuro2A cells respectively (180 ng plasmids/well) with 1.8 μl lipofectamine 2000 (Invitrogen, CA, USA). 18 h post-transfection, the cells were harvested and incubated with propidium Iodide (PI, Sigma, 50 μg/ml) and RNAase A (Thermo, 10 μg/ml) for 30 min at room temperature (Hui et al., 2013 (link)). Then, these cells were analyzed by calculating 10,000 cells per sample via flow cytometry (FlowCytometer, Beckman Coulter, Brea, CA). Cell-cycle was analyzed using Wincycle 32 software (Beckman Coulter, Brea, CA).