Dissected GR tissues of E10.5 embryos were digested in 0.25% trypsin, passed through a 0.4 μm cell strainer and suspended in DMEM/L-15 medium supplemented with 20% knockout serum replacement (Invitrogen), 2 mM l -glutamine, 0.1 mM non-essential amino acids and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), before being plated onto 0.1% gelatin-coated cover slips. Cells were incubated in 0.5% serum-containing media before treatment with 200 ng/mL recombinant SHH N-terminal peptide (R&D Systems, 1314-SH) diluted in dimethyl formamide (DMF).
For PGC co-cultures with feeder layers, single-cell suspensions generated from dissected GR tissues were plated onto the NIH3T3/Cas9 feeder layer pre-treated with Mitomycin-C (5 μg/ml). The proliferation and motility of PGCs were measured by time-lapse imaging of GFP-positive cells captured every 15 min for 10 h. Live imaging was performed using Nikon A1R laser scanning confocal microscope in a humidified 5% CO2 chamber at 37.0 ± 0.5 °C. Random motility of PGC was analyzed using the Chemotaxis and Migration Tool 2.0 plug-in software (Ibidi GmbH).
For PGC co-cultures with feeder layers, single-cell suspensions generated from dissected GR tissues were plated onto the NIH3T3/Cas9 feeder layer pre-treated with Mitomycin-C (5 μg/ml). The proliferation and motility of PGCs were measured by time-lapse imaging of GFP-positive cells captured every 15 min for 10 h. Live imaging was performed using Nikon A1R laser scanning confocal microscope in a humidified 5% CO2 chamber at 37.0 ± 0.5 °C. Random motility of PGC was analyzed using the Chemotaxis and Migration Tool 2.0 plug-in software (Ibidi GmbH).