2.9. Cell Cycle Analysis

To assess the effects of PA on the cell cycle of N2a cells, cell cycle analysis was performed by flow cytometry following staining with 4′,6-diamidino-2-phenylindole (DAPI). Cells were seeded in 12-well plates at 1.0 × 10⁵ cells/well. After overnight incubation, cells were treated with 200 µM PA or BSA in serum-free media for 6 h. At the end of treatment, cells were collected by trypsinization, rinsed with D-PBS, and then fixed in 70% ethanol on ice for at least 1 h. Ethanol-fixed cells were then spun down, rinsed with D-PBS, resuspended in D-PBS, and then incubated with 1 μg/mL DAPI staining solution for 30 min in the dark on ice. DAPI fluorescence was then analyzed by flow cytometry on the MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec; Auburn, CA, USA). Flow cytometry events were then gated, and data were analyzed using FlowJo10 (FlowJo; Ashland, OR, USA).