Encyclopedia of Experiments: Biology
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- 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 interest to isolate and analyze those cells.
- After collecting the animals, centrifuge them at 1,600 times g for five minutes. Remove all of the supernatant and re-suspend the worms in 1 milliliter of M9 media. Transfer the suspension to a 1.5-milliliter microcentrifuge tube.
Centrifuge at 1,600 times g for five minutes to pellet the worms. Then, add 200 microliters of SDS-DDT buffer and incubate at room temperature for five minutes. The worms should now appear to be wrinkled along the body if viewed under a light microscope.
Next, add 800 microliters of ice-cold isolation buffer and mix by gently flicking the tube. Centrifuge at 13,000 times g and at 4 degrees Celsius for one minute to pellet the worms. Remove the supernatant and wash with 1 milliliter of isolation buffer.
Repeat this centrifugation and wash process a total of five times, making sure to carefully remove the isolation buffer each time. After this, add 100 microliters of protease mixture from Streptomyces griseus dissolved in isolation buffer, to the pellet.
Incubate at room temperature for 10 to 15 minutes. Making sure to apply mechanical disruption by pipetting up and down 60 to 70 times with the 200 microliter micropipette tip against the bottom of the tube. To determine the stage of digestion, remove 1 to 5 microliters of the digestion mixture, drop it onto a glass slide, and inspect it using a tissue culture microscope.
After five to seven minutes, worm fragments should have a visibly reduced cuticle and a slurry of cells will be readily visible. Halt the reaction by adding 900 microliters of commercially available Leibovitz's L15 medium supplemented with 10% FBS and penicillin-streptomycin.
Centrifuge at 10,000 times g and at 4 degrees Celsius for 5 minutes to pellet isolated fragments and cells. Wash the pelleted cells twice more with cold L15 supplemented media using 1 milliliter of media per wash. Re-suspend the pelleted cells in 1 milliliter of L15 supplemented media and leave on ice for 30 minutes.
Then, take the top layer, which is approximately 700 to 800 microliters, and transfer it to a microcentrifuge tube. Use an automated cell counter or hemocytometer to measure the cell density of 10 to 25 microliters of isolated cells.
