This article details the process of isolating cerebellar granule neurons from mouse pup brains. It outlines the steps necessary to ensure a pure culture of neurons by removing contaminants such as glial cells and meninges.
Take a freshly harvested mouse pup brain.
Separate the cerebellum and remove its membranous layer.
From the ventral side, remove the network of blood vessels.
Chop the cerebellum into fragments and transfer them to a tube containing buffer.
Centrifuge and discard the supernatant containing debris.
Add trypsin, a proteolytic enzyme, to digest the tissue's extracellular matrix and loosen the cells.
Add a buffer containing trypsin inhibitor and DNase.
The inhibitor stops trypsin activity while DNase degrades any contaminating DNA.
Mechanically dissociate the tissue to form a cell suspension, including, cerebellar granule neurons and non-neuronal or glial cells.
Transfer the cell suspension to a tube.
Add buffer. Centrifuge and remove the supernatant. Resuspend cells in media and plate them onto culture plates coated with poly-D-lysine.
The cells attach to the coated surfaces.
Add an antimetabolite to eliminate proliferating glial cells and generate a pure culture of cerebellar granule neurons.
To isolate the cerebellum, place the brain in the dissection solution supplemented with magnesium sulfate, and keep the solution and the brain on ice.
Under a dissection microscope, remove the meninges using fine forceps. Then, dissect out the cerebellum from the brain in magnesium sulfate-supplemented dissection solution. This helps in peeling off the remaining meninges and allows one to go in between layers to clean the cerebellar folds.
The presence of meninges in neuronal culture results in unhealthy cells and eventual cell death. As such, it's important to ensure complete removal of the meninges before proceeding with culture.
After that, turn the cerebellum to its ventral side and ensure the removal of the choroid plexus. Next, pool the cerebella into a 35-millimeter dish containing 1 milliliter of magnesium sulfate-supplemented dissection solution. Chop the tissues into small pieces, and transfer them into a 50-milliliter tube containing 30 milliliters of magnesium sulfate-buffered dissection solution.
In this step, centrifuge the 50-milliliter tube containing the chopped cerebral tissue for five minutes at 644 times g and 4 degrees Celsius. After that, remove the supernatant, and add 10 milliliters of trypsin dissection solution. Then, shake the table at high speed for 15 minutes at 37 degrees Celsius.
Add 10 milliliters of trypsin inhibitor solution-I to the tube and rock gently for two minutes. Subsequently, centrifuge the tube for five minutes at 644 times g and 4 degrees Celsius. After five minutes, remove the supernatant, and add 2 milliliters of trypsin inhibitor solution-II before transferring it to a 15-milliliter tube.
Then, triturate the tissue in the 15-milliliter tube until the solution becomes murky. Let it settle for five minutes. Then remove the clear supernatant and transfer it to a new tube containing 1 milliliter of calcium chloride-supplemented dissection solution.
Add another 2 milliliters of trypsin inhibitor solution-II to the bottom of the tube containing the pellet. Triturate it again and let it settle for five minutes. Remove the supernatant and add it to the tube containing supernatant from the previous step. Repeat this process until most of the tissue is mechanically dissociated.
Add 0.3 milliliters of calcium chloride-supplemented dissection solution to the supernatant collection for every milliliter of supernatant. Mix the contents of the tube and then centrifuge for five minutes at 644 times g at room temperature. Following that, remove the supernatant. Add 10 milliliters of fresh media to the pellet and mix.
Then, count the live cells and dilute them to a concentration of 1.5 x 106 cells per milliliter. Plate the cells on the previously prepared poly-D-lysine plates. For four-well plates, place 0.5 milliliters of the sample, giving 7.5 x 105 cells per well. For 35-millimeter dishes, plate 4 milliliters of the sample, giving 6 x 106 cells per plate. For coverslips, plate 0.5ml, giving 7.5 x 105 cells per well.
After 24 hours, add AraC two the plates to reduce glial contamination. If the cells are to be maintained for seven to eight days, repeat this treatment on day 3, and maintain the cultures in a 5% CO2 incubator at 37 degrees Celsius.
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