Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Experimental Preparation

NOTE: The following stock solutions can be prepared and maintained until use.

1. Dissection Solution​
   1. Dissolve 3.62 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.069 g of sodium phosphate (NaH₂PO₄∙ H₂O), and 1.306 g of D-(+)-Glucose in 500 mL of distilled H₂O. Then add 12.5 mL of HEPES Buffer, 450 µL of Phenol red solution, and 20 mL of BSA fraction V to the solution. Adjust to pH 7.4 using 1 N sodium hydroxide (NaOH).
   2. Sterilize with a 0.22 µm filter and store at 4 °C. This solution will remain stable for up to one week to 10 days.
2. Trypsin
   1. Prepare a stock solution at a concentration of 25 g/L of trypsin in 0.9% sodium chloride. Store the solution at -20 °C.
3. Trypsin Inhibitor
   1. Prepare a stock with a concentration of 10 mg/mL by dissolving 1 g of chicken egg white trypsin inhibitor in 100 mL of 1 mM HCl. Store this solution at -20 °C.
4. DNase​
   1. Dissolve 100 mg of DNase 1 in 10 mL of sterile water to generate a 10 mg/mL stock. Store the solution at -20 °C.
5. MgSO₄
   1. Add 6.02 g of magnesium sulfate (MgSO₄) to 50 mL of distilled water (H₂O) to generate a 1 M stock. Store the solution at 4 °C.
6. CaCl₂
   1. Dissolve 0.735 g of calcium chloride (CaCl₂∙ 2H₂O) in 50 mL of distilled H₂O to a stock concentration of 100 mM. Store solution at 4 °C.
7. KCl​
   1. Dissolve 3.7275 g of KCl in 50 mL of distilled H₂O to a stock concentration of 1 M. Store solution at 4 °C.
8. D-(+)-Glucose
   1. Dissolve 1.802 g of D-(+)-glucose in 50 mL of distilled H₂O to a stock concentration of 100 mM. Store the solution at 4 °C.
9. Cell Culture Media
   1. Prepare cell culture media as follows: 5 mL of heat-inactivated dialyzed Fetal Bovine Serum, 500 µL of 200 mM L-Glutamine, 100 µL of 50 mg/mL Gentamycin and 1 mL of 1.0 M KCl should be added to 45 mL of filter sterilized Eagle's minimal essential media (E-MEM) that is supplemented with 1.125 g/L D-Glucose to generate 50 mL of cerebellar granule neuron culture media. Store media at 4 °C.
10. Cytosine Beta-D- Arabino Furanoside​
    1. Dissolve 48 mg of cytosine beta-D-arabinofuranoside in 10 mL of growth media to a stock concentration of 20 mM. Store solution at -20 °C.
11. Poly-D-Lysine
    1. To generate a 2 mg/mL poly D-lysine stock, add 10 mL of sterile H₂O to 20 mg of poly D-lysine. Stock poly D-lysine should be stored at -80 °C in 100 µL aliquots.
       NOTE: The following solutions should be prepared prior to dissection and maintained at 4 °C until use.
12. MgSO₄ Supplemented Dissection Solution
    1. Add 300 µL of stock MgSO₄ to 250 mL of dissection solution.
13. Trypsin Dissection Solution
    1. Add 100 µL of stock trypsin and 12 µL of stock MgSO₄ to 10 mL of dissection solution.
14. Trypsin Inhibitor Solution 1​
    1. Add 164 µL of stock trypsin inhibitor, 250 µL of stock DNase 1 and 12 µL of stock MgSO₄ to 10 mL of dissection solution.
15. Trypsin Inhibitor Solution 2
    1. Add 1040 µL of stock trypsin inhibitor, 750 µL of stock DNase 1 and 12 µL of stock MgSO₄ to 10 mL of dissection solution.
16. CaCl₂ supplemented dissection solution
    1. Add 10 µL of stock CaCl₂ and 25 µL of stock MgSO₄ to 10 mL of dissection solution.
17. Poly D-Lysine Coated Culture Dishes​
    1. To coat culture dishes, dilute 100 µL of stock poly D-lysine in 40 mL of sterile H₂O. For 35 mm Nunc culture dishes, add 2 mL of diluted poly D-Lysine solution. For 4 well plates, add 300 µL of diluted poly D-Lysine to each well.
    2. Let the poly D-lysine sit for 1 h before aspirating and washing each well with 4 mL or 600 µL (for 35 mm culture dishes or 4 well plates, respectively) of sterile H₂O. Then aspirate the H₂O and let the dishes air dry for 1 h.

2. Brain Extraction and Isolation of Cerebellum

1. Decapitate a 6-7 day-old mouse pup using decapitation scissors. Perform the dissection of the brain in a sterile tissue culture hood.
2. To remove the brain, grasp the head using a pair of forceps and cut through the skin toward the anterior of the head using microdissection scissors, as demonstrated in Figure 1. Then cut through the skull bone using a new pair of scissors to minimize the risk of contamination. Remove the skull covering the brain using forceps and tease out the brain using forceps or a spatula.​
   1. As needed, cut through the optic nerve to facilitate getting the brain out as a whole.
3. Place the brain in the dissection solution which is supplemented with MgSO₄. Keep the solution and the brain on ice.
4. Following brain removal, under a dissection microscope, dissect out the cerebellum from the brain while still in MgSO₄ supplemented dissection solution, and remove the meninges using fine forceps. Turn the cerebellum to its ventral side and insure removal of the choroid plexus.
5. Pool the cerebella into a 35-mm dish containing ~1 mL of MgSO₄-supplemented dissection solution.
6. Chop the tissue into small pieces and transfer it into a 50 mL tube containing 30 mL of MgSO₄ buffered dissection solution.

3. Mouse Cerebellar Granule Neuron Isolation and Culturing

1. Centrifuge the 50 mL tube containing the chopped cerebral tissue for 5 min at 644 x g and 4 °C.
2. Remove the supernatant and add 10 mL of trypsin dissection solution. Then shake the tube at high speed for 15 min at 37 °C.
3. Add 10 mL of trypsin inhibitor solution 1 to the tube and rock gently for 2 min.
4. Centrifuge the tube for 5 min at 644 x g and 4 °C.
5. Remove the supernatant and add 2 mL of trypsin inhibitor solution 2, and then transfer to a 15 mL tube.
6. Triturate the tissue in the 15 mL tube until the solution becomes murky. Then let settle for 5 min.
7. Remove the clear supernatant and transfer the supernatant to a new tube containing 1 mL of CaCl₂ supplemented dissection solution.
8. Add another mL of trypsin inhibitor solution 2 to the bottom of the tube containing the pellet from step 3.6. Triturate again and let settle for 5 min. Remove the supernatant and add it to the tube containing the supernatant from step 3.7 (That is a repeat of steps 3.6-3.7). Repeat this process until most of the tissue is mechanically dissociated.
9. Add 0.3 mL of CaCl₂-supplemented dissection solution to the supernatant collection for every mL of supernatant.
10. Mix the tube contents and then centrifuge for 5 min at 644 x g.
11. Remove the supernatant and add 10 mL of fresh media to the pellet and mix.
12. Cells may then be counted and diluted to a concentration of 1.5 x 10⁶ cells/mL. Please note that in general, 10 million cells are expected from each dissected brain, dependent on trypsinization time and efficiency of dissection. Plate cells on previously made poly D-lysine plates. For 4-well plates, plate 0.5 mL, giving 7.5 x 10⁵ cells per well. For 35-mm dishes, plate 4 mL, giving 6 x 10⁶ cells per plate.
13. After 24 h, add cytosine-β-arabino furanoside (AraC) to the plates to reduce glial contamination. For each mL of media 0.5 µL of 20 mM AraC is required. Addition of AraC does not require a media change. If cells are to be maintained for 7-8 days, repeat this treatment on day 3.
14. Maintain cultures in a 5% CO₂ incubator at 37 °C and feed with 100 µL of 100 mM glucose added to the culture for every 2 mL of media every 2 days past 5 days. A complete media change is not required.