This article describes a method for isolating cerebellar granular progenitors (CGNPs) from mouse pup cerebella. The protocol involves enzymatic digestion, mechanical dissociation, and cell culture techniques to obtain and proliferate CGNPs.
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.
Transfer three to five cerebella into 15-milliliter tubes. Wash cerebella with HBSS glucose before centrifugation. Centrifuge the tubes to collect the tissue. After the final wash, homogenize the cerebella by gently pipetting up and down two to three times with a 1-milliliter pipette until the fragments are 0.5 to 1 cubed millimeters in size.
Gently remove all liquid until 2.5 milliliters are left. Then, add 0.05% trypsin into the tube with HBSS glucose containing the cerebella, and incubate the tissue in a 37 degrees Celsius water bath for 15 minutes. Invert the tissue every 1 to 3 minutes. Following the incubation, stop the digestion by adding 5 milliliters of cGMP cell culture medium or CGM, and collect the tissue by centrifugation as before.
After centrifuging, remove the supernatant and add 1 milliliter CGM. Triturate the tissue with the 1-milliliter pipette tip, avoiding the formation of air bubbles. Then, add 5 milliliters of CGM, and incubate the mixture for two minutes on ice to settle tissue remnants. Once the tissue has settled, transfer the supernatant into a new 15-milliliter tube. Then, add 2 milliliters of CGM to the residual tissue, and repeat the trituration procedure before harvesting the supernatant and discarding the tissue remnants.
Pool the supernatants from each 15-milliliter tube of tissue, and centrifuge to collect the cerebellar cells. Resuspend the pellet in 10 milliliters of CGM. Since astrocytes adhere faster and stronger to poly-D-lysine than cGMPs, add up to 4 milliliters of cell suspension to the wells of poly-D-lysine-coated 6-well plates, and incubate for 20 minutes at 37 degrees Celsius to remove astrocytes.
Shake the plate. Collect the supernatant in a 15-milliliter tube, and then centrifuge the supernatant as before. Resuspend the pellet in 10 milliliters of CGM, and count the cells with a Neubauer counting chamber. After 4 to 6 hours, the adhered cGMPs appear round, and are proliferative. Seed the cells in CGM on poly-L-ornithine-coated plates, and incubate at 37 degrees Celsius, 5% CO2, and 100% relative humidity.
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