Preparing a Single-Cell Suspension of Immune Cells from Murine Brain Tissue
Transcription
Pour the brain or spinal cord tissue with the media into a 100-millimeter Petri dish, and use forceps to move the tissue to the bottom. Mince the brain tissue with a sterile razor blade, and gather the tissue at the bottom of the plate. Add 3 milliliters of RPMI supplemented with 10% FCS to the Petri dish, then use a 10-milliliter serological pipette to resuspend the tissue in the media and transfer it to a 15-milliliter conical tube.
Add collagenase type I and DNase I to the tissue and incubate the tubes in a 37 degrees Celsius water bath for 40 minutes. Invert the tubes every 15 minutes to thoroughly mix the tissue with the enzymes. After incubation, add EDTA to each tube for a final concentration of 0.01 molar, and incubate the tubes for an additional five minutes to inactivate the collagenase.
Add 9 milliliters of RPMI, supplemented with 10% FCS to each tube, then, centrifuge the tubes at 450 g for 5 minutes at 4 degrees Celsius. Use a Pasteur pipette with a vacuum to aspirate the supernatant, taking care not to touch the cell pellet.
Add 3 milliliters of 100% stock isotonic density gradient solution to the cell pellet. Then, add additional RPMI 10% FCS media to bring the final volume to 10 milliliters and resuspend the cell pellet. Invert and mix the tube well. Then, insert a serological pipette with 1 milliliter of 70% stock isotonic density gradient solution into the bottom of the tube.
Slowly underlay the solution, being careful not to make bubbles. Remove the serological pipette from the tube, making sure not to disturb the gradient. Centrifuge the tube at 800 g for 30 minutes at 4 degrees Celsius, then aspirate the supernatant, including the myelin debris layer until 2 to 3 milliliters remain in the tube.
Use a 1-milliliter pipette to harvest the cell layer between the 30% and 70% density gradient and transfer it to a new 15-milliliter tube. Add RPMI 10% FCS media to bring the final volume to 15 milliliters, then centrifuge the cells at 450 g for 5 minutes at 4 degrees Celsius.
