Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors
Transcription
Take mouse pup cerebella.
Homogenize them into smaller fragments.
Add a proteolytic enzyme to digest the tissue’s extracellular matrix, loosening cerebellar granular progenitors, or CGNPs, and astrocytes.
Add CGNP culture media containing serum to stop the enzymatic activity. Centrifuge and discard the enzyme-rich supernatant.
Resuspend the tissue fragments in CGNP media. Mechanically dissociate the tissue to release the cells.
Once the debris settles, transfer the supernatant containing the cells to a fresh tube.
Centrifuge and remove the supernatant with debris.
Resuspend the cells in CGNP media, and seed them onto multi-well plate wells coated with poly-D-lysine. Incubate.
Astrocytes settle and adhere strongly to the poly-D-lysine coating, compared to CGNPs.
Shake the plate and collect the supernatant containing CGNPs. Centrifuge and remove the supernatant.
Resuspend CGNPs in CGNP media and seed them onto a poly-L-ornithine-coated multi-well plate. Incubate.
CGNPs attach to the coated surface and proliferate, aided by the media components and poly-L-ornithine.
