Isolation of Cells from Sensory Ganglia
Transcription
After harvesting the sensory ganglia, aspirate 500 microliters of ganglia dissociation solution from each tube without disturbing the ganglia. Next, add 1 milliliter of PBS to each sample and wait for the ganglia to settle to the bottom of the tubes.
Then, remove the PBS and add 1 milliliter of ganglia dissociation solution supplemented with digestion enzyme to the neurons. Incubate the tubes at 37 degrees Celsius for the appropriate time, based on the age of the digestion enzyme, shaking and flicking the tubes briefly every 15 minutes to ensure the ganglia are covered by the digestion solution.
While the nerve cells are being digested, slowly and carefully add 400 microliters of freshly prepared 12% density solution over 400 microliters of 28% density solution in one 1.5-milliliter tube per sample. Two distinct layers should be visible, one darker than the other.
At the end of the digestion period, discard the digestion enzyme, and wash the ganglia two times with 1 milliliter of PBS as just demonstrated. After the second wash, add 200 microliters of fresh ganglia dissociation solution to each tube.
Then, using a 200-microliter pipette set to a 100-microliter volume, pipette the ganglia up and down several times to dissociate the cells into a single-cell suspension, taking care to avoid bubbles. When no intact tissue pieces are visible, holding a 70-micron cell strainer firmly in one hand, pipette the dissociated cell solution into the center of the strainer. Then, wash the dissociation tube with 100 microliters of fresh ganglia dissociation solution to collect any additional cells.
Filter the wash through the strainer, and use a new pipette tip to aspirate any remaining cell-containing solution on the underside of the strainer, adding it to the cell suspension. Now, carefully layer all 300 microliters of the cells on top of the previously prepared density gradient, and collect the ganglia cells by centrifugation.
At the end of the centrifugation, carefully remove the top 700 microliters of solution containing the majority of the cell debris. Then, mix 700 microliters of fresh ganglia dissociation solution with the remaining cells. Pellet the cells by centrifugation, and carefully discard the supernatant.
