Substrates, including human plasma fibronectin (Millipore) and BSA (Sigma), were diluted at 10 µg/ml in PBS and coated on plastic at 4°C overnight. The migration assay with CNC explants on fibronectin-coated plastic dishes was performed as previously described (DeSimone et al., 2005). For the adhesion assay, fluorescently labeled CNC explants from the embryos injected into the animal hemisphere with eGFP mRNA were incubated in Ca2+- and Mg2+-free MBS for 1 h at 12°C to dissociate the cells (Alfandari et al., 2003). The single CNC cells were subsequently plated in 96-well plates coated with fibronectin or BSA and allowed to attach for 45 min at room temperature. Photographs were taken with a fluorescence microscope shortly before and after gently pipetting the medium up and down, and the attached cells were counted.
