Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

Take a perfused rat brain. Mince it into small fragments.

Homogenize the tissue in a buffer to release cells, including microglia.

Once the undigested tissue settles, collect the supernatant containing cells and add a myelin separation buffer.

Mix the contents and centrifuge to separate cells from myelin and debris. Remove the myelin-debris layer and supernatant.

Resuspend cells in buffer and pipette to separate clumps.

Pass the suspension through a strainer to remove remaining cell aggregates.

Transfer the filtered cells to a culture plate coated with microglia-specific monoclonal antibodies. Incubate to facilitate microglia adhesion.

Wash with buffer to remove non-adherent cells. Add trypsin and incubate to loosen microglia.

Remove trypsin.

Add serum-free media. Incubate on ice to weaken cell-substrate interactions.

Pipette and collect the suspension in a tube. Centrifuge and remove the supernatant.

Resuspend microglia in serum-free media and transfer onto a collagen-coated multi-well plate. Incubate to facilitate microglia adhesion.

Add media with growth factors and lipids. Incubate for microglia maturation.

After the brains have been collected in cold DPBS, chop each brain into