Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells

- Start with washed pelleted worms and add lysis buffer containing SDS, a detergent, and DTT, a reducing agent. This breaks down the worm's protective cuticle exposing the body for cell dissociation. Avoid prolonged exposure to the lysis buffer to minimize cell lysis and death.

Next, remove the lysis reagents with several washes of cold isolation buffer. It is important that this buffer is of the correct osmolarity to prevent cell death. Use a protease enzyme to breakdown cell to cell connections. Help the homogenization process along with mechanical energy by pipetting the mixture against the tube wall.

Add media containing serum to stop the enzyme reaction and antibiotics to prevent contamination. Pellet and wash the sample several times to remove most of the debris.

Finally, place the tube on ice to allow for gravity sedimentation. Debris including remnants of the cuticle or cell clumps will settle out, while dissociated cells remain suspended in the top layer of media. These cells can be used for short term culturing or to isolate specific cell populations by FACS or immunoprecipitation.

In the example protocol, we will dissociate transgenic worms expressing GFP in neurons of i