A Simple Technique to Isolate and Culture Murine Astrocytes
Transcription
Cut a mouse cortex into small pieces and transfer to a cold dissection medium containing a buffered salt solution.
Add digestive enzymes to dissociate the extracellular matrix.
To remove residual enzymes, wash with the cold dissection medium.
Replace this medium with an astrocyte-enrichment medium.
Next, use a pipette to dissociate the tissue pieces.
Pass the suspension through a strainer to remove undissociated tissue and obtain single cells.
Transfer the cells to a positively charged polymer-coated plate. Incubate with shaking.
Astrocytes adhere firmly, while the loosely bound neurons, microglia, and oligodendrocytes detach.
The enrichment medium converts mature astrocytes into dividing polygonal astrocytes.
Remove the detached cells and wash astrocytes with a phosphate buffer.
Add digestive enzymes to detach astrocytes.
Add a medium containing serum to stop the enzymatic activity. Collect the astrocytes in a tube and centrifuge.
Discard the supernatant. Resuspend cells in an astrocyte-maturation medium to promote a typical star-shaped mature astrocyte structure.
