Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes
DEŞİFRE METNİ
The astrocyte in vitro culture helps investigate the biological role of astrocytes – the most abundant brain cells.
Begin with a neonatal mouse brain placed under a dissecting microscope.
Remove the olfactory bulbs and the cerebellum.
Perform a midline incision between the two hemispheres to separate the cortex.
Remove the meninges to avoid contamination in the astrocyte culture from fibroblasts and meningeal cells.
Transfer the cortex hemispheres into an ice-cold medium and mince into small pieces.
Collect the cortical pieces in a tube containing trypsin. Trypsin, a protease enzyme, digests the cell adhesion proteins, facilitating cell separation.
Centrifuge to pellet the digested tissue.
Remove the supernatant and add an astrocyte medium to the pellet. Pipette vigorously to release a mixed cell population of astrocytes, microglia, and oligodendrocyte precursor cells or OPCs.
Seed this cell suspension into a poly-lysine coated tissue culture flask and incubate.
The negatively charged cells attach to the positively charged poly-lysine and grow over the entire surface to form a confluent layer.
Shake the flask to detach the loosely bound microglia and OPCs. Remove the supernatant containing detached cells.
Add trypsin to the astrocyte layer. Collect the cells and centrifuge.
Resuspend the pellet and divide the astrocytes into multiple flasks.
Finally, assess the purity of the astrocyte culture by immunostaining cell-specific markers.
