Culturing Neural Crest Explants

Prior to embryo dissection, tissue cultureware (Celltreat, 229168-chambered microscope slides were used for imaging) were coated with 25 ug/ml fibronectin (Sigma, F1141) and incubated for at least 45 min in a 37 °C humidified incubator maintained at 5% CO₂ atmosphere. After incubation, the fibronectin solution was removed from the cultureware and DMEM (Sigma, D6046) supplemented with 10% FBS (Thermo Fisher Scientific, 10-437-028) was added. NCC explants were obtained by thinly micro-dissecting dorsal neural folds from HH9 (6–7 somite) embryos in Ringer’s solution as previously described^{18 (link)}. Each dissected neural fold was then aspirated in a minimal amount of Ringer’s solution (2.5 ul) and placed in a well of the fibronectin-coated tissue cultureware containing media. The explants were incubated for at least 12 h under normoxic conditions in a humidified 37 °C incubator maintained at 5% CO₂ atmosphere. The duration of incubation was determined by the appropriate conditions pertaining to specific experiments.