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

FIGURE 5.

FIGURE 5

B-27 encourages neurite outgrowth and differentiation in SH-SY5Y cells. SH-SY5Y cells were cultured in DMEM/F-12 supplemented with either 10% FBS, 2% B-27 supplement, 5% B-27 supplement or 0.5% FBS + 20 ng/ml BDNF + 10 μM RA for 5 days. 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 through the DAPI channel (excitation 325–375 nm; emission 435–485 nm) and the FITC channel (excitation 460–500 nm; emission 512–542 nm) on the Leica DM4000 LED upright fluorescent microscope. Scale bar = 100 μm. 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.