Prolong gold antifade mountant with 4 6 diamidine 2 phenylindole dihydrochloride dapi

Anterior NPCs (aNPCs) were derived from parental iPSC lines as detailed in supplementary methods. Following plate down in RosV2 F10 [Advanced-DMEM/F12, 1% P/S, 1% Glutamax, 1% N-2, 0.5% B-27, supplemented with 2.5 ng/mL FGF-2] media was changed to default [Advanced-DMEM/F12, 1% P/S, 0.5% Glutamax, 0.5% N-2, 0.2% B-27, 2 μg/mL Heparin (Sigma)] to remove the effects of mitogens. To assess NPC proliferation mitotic cells were grown to ~50% confluency (to ensure consistency in experimental conditions) and labelled with 10 μM ethynyl deoxy-uridine (Click-IT EdU, Life Technologies) for 4 h. Media was replaced completely and cells cultured for an additional 4 h in the absence of EdU before fixation using 4% paraformaldehyde (PFA) (Sigma). Cells were permeabilised using 0.5% Triton X-100 (Sigma) in PBS for 20 mins and detected using the EdU detection kit (ThermoFisher) according to manufacturer’s instructions. NPCs were also immunolabelled for Nestin and Pax-6 (see Supplementary Table 2 for antibody details). Coverslips were washed and mounted using ProLong Gold antifade mountant with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (ThermoFisher).