Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture
Trascrizione
Take a harvested rat cerebellum and chop it into fragments.
Transfer the fragments to a tube containing trypsin and incubate.
Trypsin digests the tissue's extracellular matrix, loosening the cerebellar granule neurons, as well as non-neuronal cells such as microglia and astrocytes.
Add a buffer containing a trypsin inhibitor and DNase. The inhibitor inactivates trypsin while DNase degrades contaminating DNA.
Centrifuge and discard the supernatant.
Resuspend the tissue in a buffer containing the inhibitor and DNase, and mechanically dissociate it to form a single-cell suspension.
Overlay the suspension onto a protein-containing buffer solution.
Centrifuge and discard the supernatant containing cellular debris.
Resuspend the cells in media and seed them on poly-L-lysine-coated coverslips to facilitate cellular attachment.
Remove the media. Wash the cells with fresh media, and then, add media containing a cell cycle inhibitor to prevent glial cell proliferation.
Replace half of the media with fresh media containing a lysosome inhibitor.
The inhibitor selectively targets microglia and disrupts lysosomal membranes, causing microglial death and removal from the culture.
