Isolation of Stem Cells from Postnatal Mouse Cerebellum and Differentiation into Neural Cells
Transcript
Place the centrifuge tubes on ice, then, strain the dissociated cells through a 40-micrometer cell strainer into a 50-milliliter tube. Top the filter with 10 milliliters of ice-cold HBSS solution, to ensure that the cells pass through the mesh.
Transfer the filtered cells to a fresh 15-milliliter centrifuge tube, and centrifuge the suspension at 300 times g for 10 minutes at 4 degrees Celsius. Then, use a vacuum aspirator to remove the supernatant completely.
Resuspend the cell pellet in 160 microliters of ice-cold magnetic column buffer. Add 40 microliters of anti-prominin-1 microbeads to the cell suspension. Then, incubate the tubes in a refrigerator for 15 minutes, to allow the antibody to bind to the prominin-1-expressing cells.
Wash the cells with 1 to 2 milliliters of column buffer X, and centrifuge them at 300 times g for 10 minutes. Aspirate the supernatant and resuspend the pellet in 1 milliliter of column buffer X. Place the magnetic columns on the magnetic stand, and rinse them once with 500 microliters of buffer X. Apply the labeled cell suspension onto the column, and collect the flow-through in fresh 15-milliliter tubes.
Wash the column three times with 500 microliters of buffer X. Then, remove the columns from the magnetic field and place them in 1.5-milliliter tubes. Add 1 milliliter of culture medium, and push the plunger into the column to flush out the cells tagged with prominin-1 beads.
To passage the neurospheres, transfer them along with the culture medium into a sterile 15-milliliter centrifuge tube. Pellet the neurospheres by centrifugation at 300 times g for 5 minutes, and discard the supernatant.
Resuspend the pellet in 5 milliliters of dissociation media with papain or 0.05% trypsin solution, and incubate the cells at 37 degrees Celsius for 10 minutes. Centrifuge the cell suspension again. Then, resuspend the cells in 5 milliliters of neurosphere medium, and dissociate them by slowly pipetting them up and down 10 times with a pasture pipette. Plate the cells in neurosphere media as described in the text protocol, and enrich them with secondary neurospheres after 7 to 10 days in culture as previously described.
