Enzymatic Digestion and Manual Dissociation: A Method to Prepare C. elegans Embryos for Cell Culture

- Start with pelleted eggs that were isolated by bleaching gravid adult worms. Ensure each step of this procedure is conducted sterilely to avoid contamination of the cultured cells. Suspend the pellet in a solution of chitinase, an enzyme that breaks down chitin-- a large polysaccharide that is a structural component of the egg shell.

After an appropriate digestion period, gently centrifuge the eggs and replace the supernatant with cell culture medium to stop the digestion. Then, pass the embryos through an 18-gauge needle to mechanically separate individual cells. Next, filter the solution to remove debris from the egg shell or unseparated cell clumps. Plate the cells in a culture dish on a glass cover slip coated with peanut agglutinin-- a lectin that helps the cells attach to the cover slip by binding cell surface carbohydrates.

In the example protocol, we will prepare embryonic cells for in vitro morphological differentiation and analysis.

- While working under a laminar flow hood to avoid introducing bacterial contamination, resuspend the pelleted eggs in one milliliter of two milligrams per milliliter chitinase and transfer them to a fresh 15 milliliter conical tube. Rock th