Egta am

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.

The following antibodies were used for cell stimulation: purified anti-mouse CD38 (Clone 90; BioLegend, 102702), purified rat IgG2a, κ isotype control (Clone RTK2758; BioLegend, 400565). In certain experiments, the following reagents/inhibitors were used at the indicated concentration in cell culture: LPS (E. Coli Serotype R515; Enzo Life Sciences, ALX-581-007-L001), NAADP-AM (3 μM; AAT Bioquest, 21000), GSK2578215A (LRRK2 inhibitor, 1 µM; Tocris Bioscience, 4629), GPN (200 µM; Santa Cruz Biotechnology, sc-252858), EDTA (2 mM; Sigma-Aldrich, E6758), thapsigargin (500 nM; Tocris Bioscience, 1138), 8-Br-cADPR (100 µM; BIOLOG Life Science Institute, B065), 8-Br-ADPR (100 µM; BIOLOG Life Science Institute, B051), Ned-19 (100 µM; Cayman Chemical, 17527), cyclosporine (10 µM; R&D Systems, 1101/100), FK506 (5 nM; Eton Bioscience Inc, 1100060052), CHIR 99021 (GSK3B inhibitor, 5 µM; Tocris Bioscience, 4423), BAPTA-AM (10 µM; R&D Systems, 2787), EGTA-AM (10 µM; AnaSpec Inc, AS-84100), GSK2606414 (EIF2AK3/PERK inhibitor, 10 µM; Tocris Bioscience, 5107), and dorsomorphin (5 µM; Tocris Bioscience, 3093).