Isolating Microglia from Mouse Brain Demyelinating Lesions Using Magnetic Activated Cell Sorting

Dissect stained demyelinating lesions from the corpus callosum of a mouse brain.

These lesions contain brain-resident microglia expressing the cell-surface integrin CD11b. 

Collect the tissue and add a solution containing a proteolytic enzyme and DNase.

The enzyme degrades the extracellular matrix, while DNase degrades free DNA.

Add cold buffer and filter the suspension through a strainer to remove cellular clumps.

Centrifuge, discard the supernatant, and resuspend the pelleted cells in a density gradient medium.

Overlay with buffer and centrifuge to collect remaining debris in the upper layers; intact cells settle at the bottom.

Remove the debris and resuspend the cells in a buffer.

Add anti-CD11b magnetic beads to label the microglia.

Load the cells onto a column containing ferromagnetic spheres placed in the magnetic field of the magnetic separator.

Under the external magnetic field, the bead-bound microglia are retained in the column while unbound cells flow through.

Remove the column from the magnetic field. Add a buffer to elute the microglia.