Begin by adding 2.125 milliliters of isotonic density gradient to a 15 milliliter polypropylene tube containing murine brain homogenate. Top up each tube with fax buffer to a final volume of 8.5 milliliters. Gently invert the tubes 20 times to mix thoroughly.
Using a narrow graduated transfer pipette, gently underlay four milliliters of pink, 37%density gradient establishing two clean layers. Switch transfer pipettes and underlay two milliliters of the blue, 70%density gradient below the 37%layer. Transfer the tubes to a centrifuge cool to four degrees Celsius and spin them at 500 G for 20 minutes with the braking ramp set to the lowest setting.
After centrifugation, discard the myelin from the top of the 15 milliliter tube using a clean transfer pipette. Carefully collect the top fragment of the density gradient into a clean 15 milliliter polypropylene tube using another transfer pipette. Next, gather all cells in the sample by slowly circling the pipette along the sides of the tube while collecting the immune enriched fragment.
Transfer the immune enriched fragment into a new 15 milliliter polypropylene tube. Wash the sample by adding 10 milliliters of fax to the tube. Gently invert the tube 20 times to mix thoroughly.
Spin the tubes in a cooled centrifuge with the braking ramp set to the lowest setting to pellet the isolated immune cells.
