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. 2022 Oct 20;13:943627. doi: 10.3389/fphar.2022.943627

FIGURE 7.

FIGURE 7

SH-SY5Y cells differentiated with B-27 show no significant difference in neurite length compared with SH-SY5Y cells differentiated with BDNF. (A) SH-SY5Y cells were differentiated for 5 days with DMEM/F-12 + 10 μM RA, in conjunction with varying combinations of neurite outgrowth-stimulating molecules (B-27 or BDNF) or serum starvation (0.5% FBS). Cells were fixed with 4% PFA for 20 min. Microtubules were stained with 1:500 mouse anti-β(III)-Tubulin (R&D Systems, #MAB1195) and visualized with 1:400 Alexa Fluor® 488-conjugated donkey anti-mouse (Thermo Scientific, #A21202) (green). Nuclei were counterstained with 1:5,000 DAPI (blue). Images taken at ×20 magnification. Scale bar = 100 μm. (B) Longest neurite length and total neurite length measurements were taken using the FIJI software (Schindelin et al., 2012). Three independent experimental replicates were performed and 80 cells per condition were measured in total. Data presented as mean ± SEM. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using ordinary one-way ANOVA and Tukey’s multiple comparison tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com. ns = not significant. BDNF, brain-derived neurotrophic factor; DAPI, 4′,6-Diamidino-2-phenylindole; DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PFA, Paraformaldehyde; RA, retinoic acid.