2.10. Quantitative Analysis of Neurite Outgrowth

Neurite outgrowth of Tet-On Aβ₄₂-GFP SH-SY5Y cells was measured with a Sholl analysis [24]. The cells were plated in 6-well plates at a density of 5 × 10⁴ cells/well with 10 μM RA, pretreated with 10 μM wogonin for 24 hours, and then induced with 10 μg/mL Dox for five days. The cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, which were followed by staining with 0.25% (w/v) crystal violet in 2% ethanol/water for 30 min at room temperature. The samples were observed with a microscope, and neurite outgrowth was analyzed as the length of the neurite, while neurite complexity was determined by the number of intersections of the neurites and concentric circles.