Neuro-2A Cell Differentiation Assay

Neuro-2A cells, which are mouse neuroblastoma-derived clonal cells, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and antibiotics (100 U/mL penicillin, and 100 µg/mL streptomycin) with 5% CO₂ at 37 °C. The serum was stripped of hormones by constantly mixing with 5% (w/v) AGXI-8 resin (Bio-Rad, Hercules, CA, USA) and powdered charcoal prior to ultrafiltration [48 (link)]. Neuro-2A cells were seeded at a density of 1 × 10⁵ cells/1 mL per well the day before the experiment in DMEM + 10% FBS on 6- or 24- well plates coated with poly-l-lysine. Differentiation was induced by serum deprivation as described previously [49 (link),50 (link)]. In short, the DMEM +10% FBS culture medium was changed to a DMEM +1% FBS culture medium to induce differentiation. On the next day, the culture medium was changed to prewarmed DMEM containing 1% FBS with or without the indicated concentration of S-equol, 10 nM of G15 and/or 10 nM of ICI to trigger differentiation, and it was then cultured for one to three days. The cultures were then harvested for RT-qPCR and immunocytochemistry. The cells were rinsed three times with PBS, fixed with 4% PFA, and then blocked with 2% FBS. The cells were then incubated with mouse monoclonal anti-β-tubulin III (neuronal) antibody and rabbit anti-doblecortin (C-terminal) antibody (1:200; Sigma) followed by donkey anti-mouse IgG (H + L) secondary antibody, Alexa Fluor^® 594 and donkey anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor^® 488 conjugate (1:200; Thermo Fisher Scientific, Inc.). Cell nuclei were also stained with DAPI. The cells were then inspected under a laser confocal scanning microscope (Zeiss LSM 880, Carl Zeiss Microscopy GmbH). Neurite length measurements were performed using ImageJ Fiji (NIH).