A new method for isolating primary mesenchyme cells of the sea urchin embryo. Panning on wheat germ agglutinin-coated dishes.
This paper describes a rapid and efficient way to isolate primary mesenchyme cells (PMCs) of the sea urchin embryo. The procedure involves three simple steps: Dissociation of mesenchyme blastulae in calcium-free artificial seawater. Incubation of the resulting cell suspension on dishes that have been coated with wheat germ agglutinin (WGA), to which the PMCs adhere more firmly than do other cell types. Gentle rinsing of the dishes to remove loosely attached cells, followed by more vigorous rinsing to remove PMCs. This panning procedure has been applied to embryos of three species of sea urchins, Lytechinus variegatus, L. pictus and Arbacia punctulata, and yields populations of PMCs that are 95-99% pure as determined by the proportion of cells that stain with fluorescein isothiocynate (FITC)-WGA and with a monoclonal antibody that binds specifically to PMCs. The yield of PMCs is 4-5 X 10(6) cells/100-mm dish, or 1-2 X 10(7) PMCs/ml of packed embryos. The principal advantages of this procedure are that it can be carried out rapidly and simply, and it yields pure populations of PMCs.
D000818:Animals; D000911:Antibodies, Monoclonal; D001755:Blastocyst / Q000166:cytology*; D002469:Cell Separation / Q000379:methods; D004625:Embryo, Nonmammalian / Q000166:cytology*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D012617:Sea Urchins / Q000196:embryology*; D014909:Wheat Germ Agglutinins
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D014909:Wheat Germ Agglutinins; C047939:fluorescein isothiocyanate-wheat germ agglutinin; D016650:Fluorescein-5-isothiocyanate