Enzymatic Digestion and Manual Dissociation: A Method to Prepare C. elegans Embryos for Cell Culture
Trascrizione
– Start with pelleted eggs that were isolated by bleaching gravid adult worms. Ensure each step of this procedure is conducted sterilely a avoid contamination of the cultured cells. Suspend the pellet di 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 a stop the digestion. Then, pass the embryos through an 18-gauge needle a mechanically separate individual cells. Next, filter the solution a remove debris from the egg shell or unseparated cell clumps. Plate the cells di a culture dish on a glass cover slip coated with peanut agglutinin– a lectin that helps the cells attach a the cover slip by binding cell surface carbohydrates.
In the example protocol, we will prepare embryonic cells for di vitro morphological differentiation and analysis.
– While working under a laminar flow hood a avoid introducing bacterial contamination, resuspend the pelleted eggs di one milliliter of two milligrams per milliliter chitinase and transfer them a a fresh 15 milliliter conical tube. Rock the tube for 10 a 30 minutes at room temperature, depending on the freshness of the enzyme. When approximately 80% of the egg shells are digested, centrifuge the eggs at 900 g for three minutes.
After carefully removing the supernatant, add three milliliters of L15 medium. Transfer the eggs into a six- centimeter diameter plate and begin manual dissociation using a 10-milliliter sterile syringe with an 18-gauge needle. To monitor the degree of dissociation, place a drop of suspension into a fresh Petri dish and view under a microscope. Continua until approximately 80% of the cells are dissociated.
To remove cell clumps, undigested eggs, and hatched larvae, gently filter the suspension through a five- micron filter and run an additional four a five milliliters of L15 medium through the filter a recover the cells. To culture the cells after centrifuging the filtrate at 900 g for three minutes, re-suspend the cells in complete L15 medium. Plate one milliliter per well, and store plates di a humid sealed chamber at 20 degrees Celsius di ambient air.
