Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex
Transcrição
Take a mouse pup cortex.
Add a proteolytic enzyme that digests the tissue's extracellular matrix. Then, mechanically dissociate the tissue, loosening the cells.
Add glial media and DNase to degrade any contaminating DNA.
Mechanically dissociate the tissue to release neurons and glial cells.
Once the undigested tissue settles, collect the media containing the cells and centrifuge. Remove the supernatant.
Resuspend cells in glia media and plate them on an extracellular matrix protein-coated flask.
Incubate. Astrocytes adhere more strongly to the coated surface than other cells. Glial media promote only glial cell survival.
Replace the media to remove debris.
Incubate with shaking to detach the overlying oligodendrocyte precursor cells, OPCs, and microglia.
Transfer the media with the cells onto a culture plate.
Incubate with shaking to facilitate differential microglial adhesion.
Transfer the non-adherent OPC supernatant onto a poly-D-lysine-coated multi-well plate. Incubate for OPC adherence.
Replace the media with OPC media, allowing OPC growth.
