Chemotaxis Assay for Neural Crest Cells

For chemotaxis assays, migration of explants to Sdf-1 was assessed using a bead assay (Theveneau et al., 2010 (link)). This was done by incubating heparin-acrylic beads (Sigma-Aldrich) overnight at 4°C in PBS supplemented with 1 µg/ml Sdf-1 and placing the beads ∼1 mm apart in a line of silicone grease (VWR) on fibronectin-coated dishes. Explants were then plated perpendicularly at a distance of 250–500 µm. To test the effects of Ca²⁺ buffering, explants were incubated for 30 min with 50 µM BAPTA-AM (Cambridge Bioscience) or EGTA-AM (AnaSpec). Time-lapse imaging was performed in Danilchick’s medium using an upright microscope (Eclipse 80i; Nikon) fitted with an objective (Plan Fluor 10×/0.30 DIC L/N1) and a camera (ORCA-05G; Hamamatsu Photonics). Data were acquired using SimplePCI software. Tracking of migrating neural crest cells was performed using the ImageJ Manual Tracking plug-in. Immunocytochemistry was performed using a primary rabbit antibody to phosphopaxillin Tyr118 (1:200 dilution; EMD Millipore; Theveneau et al., 2013 (link)). Explants were costained with 2 µg/ml phallodin and 2 µg/ml DAPI.