Isolation of Microglia from the Cortex of an Adult Mouse Brain

First, use a razor blade to chop each brain region into fine pieces that are less than 1 cubic millimeter. Using a 1-milliliter pipette with the tip cut off, transfer the tissue pieces into pre-chilled Dounce homogenizers. Homogenize the tissue by slowly twisting the piston in and out of the Dounce homogenizer for 6 to 10 full strokes until no visible chunks are present. Then, transfer the dissociated tissues into 50-milliliter tubes through 70-micrometer strainers.

Rinse each Dounce homogenizer and piston with a total of 6 milliliters of cold medium A, then transfer the rinsing to a 15-milliliter tube for centrifugation. Centrifuge single-cell suspension at 400 times g and at 4 degrees Celsius for 5 minutes with a break at 5.

Rinse one large depletion column and three large selection columns in a magnetic separator with 3 milliliters of MCS. When the centrifugation for the tissue samples is complete, pipette out and discard the supernatant without disturbing the pellet. Resuspend the cells in MCS buffer that contains RNase inhibitor.

Add 100 microliters of myelin removal beads to each tube containing resuspended cells from the cortex and cerebellum, and add 50 microliters of myelin removal beads to each of the tubes containing resuspended cells from the hippocampus and striatum. Incubate the tubes on ice for 10 minutes.

After this, add MCS to the tube containing cortical cells to bring its volume up to 2 milliliters, and to the remaining tubes to bring each of their volumes up to 1 milliliter. Once the columns are empty of rinsing buffer, load 2 milliliters of cortical cells onto the large depletion column and 1 milliliter of each other cell suspension onto separate large selection columns. Next, wash the large depletion column once with 1 milliliter of MCS buffer, and wash each large selection column twice using 1 milliliter of MCS buffer for each wash.