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