Single Cell Dissociation of Caenorhabditis elegans: A Method to Isolate Live Cells
Transcrição
– 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 para the lysis buffer para 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 para prevent cell death. Use a protease enzyme para breakdown cell para cell connections. Help the homogenization process along with mechanical energy by pipetting the mixture against the tube wall.
Add media containing serum para stop the enzyme reaction and antibiotics para prevent contamination. Pellet and wash the sample several times para remove most of the debris.
Finally, place the tube on ice para allow for gravity sedimentation. Debris including remnants of the cuticle or cell clumps will settle out, while dissociated cells remain suspended de the top layer of media. These cells can be used for short term culturing or para isolate specific cell populations by FACS or immunoprecipitation.
In the example protocol, we will dissociate transgenic worms expressing GFP de neurons of interest para 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 de 1 milliliter of M9 media. Transfer the suspension para a 1.5-milliliter microcentrifuge tube.
Centrifuge at 1,600 times g for five minutes para pellet the worms. Then, add 200 microliters of SDS-DDT buffer and incubate at room temperature for five minutes. The worms should now appear para 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 para 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 para carefully remove the isolation buffer each time. After this, add 100 microliters of protease mixture from Streptomyces griseus dissolved de isolation buffer, para the pellet.
Incubate at room temperature for 10 para 15 minutes. Making sure para apply mechanical disruption by pipetting up and down 60 para 70 times with the 200 microliter micropipette tip against the bottom of the tube. To determine the stage of digestion, remove 1 para 5 microliters of the digestion mixture, drop it onto a glass slide, and inspect it using a tissue culture microscope.
After five para 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 para 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 de 1 milliliter of L15 supplemented media and leave on ice for 30 minutes.
Then, take the top layer, which is approximately 700 para 800 microliters, and transfer it para a microcentrifuge tube. Use an automated cell counter or hemocytometer to measure the cell density of 10 para 25 microliters of isolated cells.
