Isolation of Microglia from the Cortex of an Adult Mouse Brain

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Mechanical Tissue Dissociation

NOTE: Keep cells and reagents cool all the time except during staining steps. This step should take ~30 min.

1. Chop each brain tissue with a razor blade into <1 mm³ fine pieces.
2. Use 1 mL pipette (with tips cut off) to transfer tissue pieces into the pre-chilled Dounce homogenizers, each containing 2 mL of medium A with DNase and RNase inhibitor.
3. Homogenize the tissue by slowly twisting the piston in and out of the Dounce homogenizer for 6−10 full strokes until no visible chunks are present.
   NOTE: Douncing kills neurons and most other glial cells but leaves microglial cells rather intact. Both insufficient and over-douncing can lead to a low yield of cells.
4. Transfer dissociated tissues into 50 mL tubes through 70 µm strainers.
5. Rinse each Dounce homogenizer and piston with a total of 6 mL of cold medium A and filter the rinsing solution through the same strainer into the corresponding tube.
6. Transfer the single cell suspensions (about 8 mL each) into 15 mL conical tubes and centrifuge at 400 x g for 5 min at 4 °C with brake = 5.

2. Myelin Removal

NOTE: This step should take ~60 min.

1. Prepare 1 large depletion (LD) column (for the cortex) and 3 large selection (LS) columns (for the other 3 regions) in a magnetic separator (Table of Materials). Rinse each column with 3 mL of MCS buffer.
2. Once centrifugation is finished, pipette and discard the supernatant without disturbing the pellet. For the cortex and cerebellum tissues, resuspend cells in 850 µL of MCS buffer with 1.8 µL of RNase inhibitor. For hippocampus and striatum tissues, resuspend cells in 400 µL of MCS buffer with 0.9 µL of RNase inhibitor.
   NOTE: The columns (LD vs. LS) and the volume used for resuspension are optimized based on the amount of myelin present in the tissue. If other brain regions are assayed, these conditions may need to be adjusted depending on the size of the tissue and how much myelin may exist. The effectiveness of myelin removal can be estimated in step 2.9.
3. Add 100 µL of myelin removal beads each into cells from the cortex and cerebellum and add 50 µL of myelin removal beads each into cells from the hippocampus and striatum.
4. Gently mix the cells with beads and incubate the tubes on ice for 10 min.
5. Bring the tube volume with cortical cells to 2 mL with MCS buffer and all others to 1 mL (i.e., add 1 mL for cortical cells; 500 µL for hippocampal and striatal cells; no need to add buffer to cerebellar cells).
6. Once columns are empty of rinsing buffer, place a 15 mL tube below each column. Load cortical cells (2 mL) onto the LD column and all others (1 mL each) onto the LS columns. Immediately use 1 mL of MCS buffer to wash the original tubes and load the solution onto the corresponding columns.
7. Wash the LD column once with 1 mL of MCS buffer and wash each LS column twice with 1 mL of MCS buffer each wash. Continue to collect the flow-through solution during washing.