Nunclon delta surface culture plate

Hindbrain regions of HH18 embryos were dissected in sterile PBS with penicillin-streptomycin (Pen-Strep, 1:100; Gibco, USA), then placed in a tube containing human embryonic stem cell medium [hESC; DMEM/F-12 1:1 with 20% KnockOut serum replacement, GlutaMax L-alanyl-L-glutamine (2 mM), non-essential amino acids (0.1 mM; all from Gibco), β-mercaptoethanol (0.1 mM; Sigma-Aldrich), Pen-Strep (1:100) and Fungizone (1:500)]. Media was next replaced with 1 ml of TrypLE Express (Gibco) to dissociate the tissue into single cells. After a manual disassociation by pipetting up and down, TrypLE was neutralized with 10:1 hESC medium and cells were passed through a 100 μm mesh strainer to detach adherent cells. Cells were cultured in hESC media at density of 1×10^5–6 cells/ml, seeded in a 48- or 96-well Nunclon Delta Surface culture plate (Thermo Fisher Scientific) and incubated at 37°C in 5% CO₂ (Peretz et al., 2016 (link), 2018 (link)). For live imaging, cell plates were imaged every 3-6 h in IncuCyte S3 Zoom HD/2CLR time-lapse microscopy system, equipped with a 20× Plan Fluorobjective (Sartorius). Time-lapse movies were generated by capturing phase images for up to 5 days of incubation (Wang et al., 2020a (link)).

Hindbrain primary cell cultures were prepared from hindbrains dissected from st.18 embryos. Hindbrains were incubated for 10 minutes at 37 °C with TrypLE Express (Gibco, USA) to dissociate the tissue into single cells, then TrypLE was neutralized with 10:1 standard cell culture medium containing DMEM/F-12 1:1 with 10 % fetal bovine serum, Penicillin-Streptomycin (Pen-Strep, 1:50; Gibco, USA) and Amphotericin B (1:400; Sigma-Aldrich, USA). Cells were passed through a 100-μm mesh strainer and centrifuged at 600 g for 10 minutes. Excess medium was carefully removed and cells were plated in a 24-well Nunclon Delta Surface culture plate (Thermo Scientific, USA) with standard medium or stem cell medium containing DMEM/F-12 1:1 with 20 % KnockOut serum replacement, GlutaMax L-alanyl-L-glutamine (2 mM), non-essential amino acids (0.1 mM; all from Gibco, USA), β-mercaptoethanol (0.1 mM; Sigma-Aldrich, USA), PenStrep (1:50) and Amphotericin B (1:400). Cell cultures were incubated at 37 °C/5 % CO₂ and culture media were replaced every 48 h. For replating experiments, cells were dissociated with TrypLE and centrifuged as described above, then plated with both types of media.

Embryonic chick hindbrains and forebrains were obtained by dissecting the relevant domains of the embryonic neural tube of st.18 HH embryos. Dissected tissues were incubated in enzyme solution (TrypLE Express, Gibco, USA) for 10 minutes (min) at 37 °C. Tissue was further dissociated into single cell suspension by manual pipetting. TrypLE was neutralized by adding standard media (1:1 DMEM/F-12, (Gibco, USA), 10% fetal bovine serum (Sigma-Aldrich USA), penicillin-streptomycin (1:50; Gibco, USA) and amphotericin B (1:400; Sigma-Aldrich, USA). Cells were passed through cell strainer (100 um) to eliminate aggregates and plated in different dilutions in a 24-well Nunclon Delta Surface culture plate (Thermo Scientific, USA) with standard media (as described above) or embryonic stem cell (SC) media (DMEM/F-12 1:1, 20% KnockOut serum replacement, 2 mM GlutaMax L-alanyl-L-glutamine, 0.1 mM non-essential amino acids (all from Gibco, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, USA), penicillin-streptomycin (1:50) and amphotericin B (1:400)). Cells were incubated in standard conditions (37 °C/5% CO₂). Unless stated differently, media was refreshed every 72 hours (hr).