Cranial Neural Crest Explants

The cranial neural crest (CNC) explant assay was originally designed to assess the basic requirements for CNC migration in vitro.This protocol describes the key parameters of CNC explants in Xenopus laevis, with a focus on how to extirpate CNC cells and assay their migration in vitro.The protocol can be adapted according to the needs of the experimenter, some examples of which are discussed here. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.RECIPES: Please see the end of this protocol for recipes indicated by .Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. ReagentsDanilchik medium containing 50 µg/mL of gentamycin Ethanol (70%) Fibronectin, bovine (Sigma) H 2 O (reverse osmosis [RODI] or distilled [dH 2 O]) Modified Barth's saline (MBS) (1×) For dissection medium, use 1× MBS containing 50 µg/mL of gentamycin. Phosphate-buffered saline (PBS) In addition, prepare PBS containing 1% (w/v) bovine serum albumin (BSA). Pork gelatin (optional for glass-bottom plates; see Step 2) Xenopus laevis eggsEquipment Dissecting microscope equipped with a gooseneck lighting system Embryo incubators at the desired temperature (between 15 ˚C and 19 ˚C for X. laevis) Eyelash knifeSelect a human eyelash with the desired thickness and curvature and thread it through a 23-gauge needle fitted on a 1-cc syringe.For safety purposes, the tip of the needle can be cut off with scissors before the threading.Fix the eyelash with a drop of nail polish or cyanoacrylate glue.