Isolation of Microglia from the Cortex of an Adult Mouse Brain
Transcription
Take a freshly harvested mouse brain cortex.
Chop the tissue into small fragments. Transfer them into a chilled homogenizer containing a buffer with RNase inhibitors and DNases.
Homogenize the tissue optimally to release cells into suspension, selectively preserving microglia due to their ability to withstand shear forces and maintain membrane viability, while eliminating neurons and other glial cells.
Further, RNase inhibitors degrade RNases, while DNases degrade any contaminating DNA.
Pass the cell suspension through a cell strainer to remove tissue debris.
Transfer the cell suspension to a tube. Centrifuge and remove the supernatant containing enzymes. Resuspend the cells in a buffer.
Add anti-myelin magnetic microbeads. These microbeads target the myelin protein, binding myelin debris and oligodendrocytes.
Load the cell-bead mixture into a depletion column comprising a matrix with ferromagnetic spheres.
Under the applied magnetic field, the microbead-bound myelin debris and oligodendrocytes are retained within the column, while microglial cells pass through.
Wash the column with buffer and collect the flow-through containing microglia.
