Cortical Neuron Isolation and Differentiation

NPCs were cultured, as mentioned earlier [76 (link)]. In brief, embryos (E14) were isolated from a Sprague–Dawley rat. Cortices were aseptically dissected from brains and dissociated to produce single-cell suspensions by gentle mechanical pipetting using fire-polished Pasteur pipettes. In the presence of epidermal growth factor (10 ng/mL) and fibroblast growth factor-2 (10 ng/mL), cells were then grown overnight in 5 mL of medium supplemented with DMEM-F12/Glutamax (Gibco, ThemoFisher, Waltham, MA, USA), penicillin–streptomycin (1%, Gibco) and B27 supplement (1%, Gibco) in 25 cm² flasks [77 (link)]. For ICC and PLA, coated coverslips (poly-D-lysine/laminin) were used to plate floating neurospheres. The transfection of neurospheres with specified plasmids was done by Lipofectamine^® 2000 reagent (Invitrogen). Geltrex^® matrix (Gibco) was used for transfection and live-cell imaging. The plated neurospheres were further grown for 48 h to attain differentiated cortical neurons in the medium containing 2% B27 supplement, 500 mM L-glutamine, and 1% penicillin–streptomycin with no growth factors. After culturing for 48–60 h, differentiated cortical neurons cultures were used for live-cell imaging and ICC.