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