Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

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

1. Preparations

1. Preparations for CGNP (cerebellar granular neuron progenitor) isolation
   1. Organize an appropriate mating of mice to generate P5-P7 animals to isolate the cerebellum (3-5 animals per ChIP and condition).
   2. Prepare HBSS/Glucose by adding 6 mg/mL glucose to the HBSS buffer.
   3. Prepare the CGNP cell culture medium (CGM) by adding 1 % (v/v) N2 supplement, 1 % (v/v) Penicillin-Streptomycin-Neomycin, 25 mM KCl and 10 % FCS to DMEM-F12. For CGNP cultivation, prepare CGM medium without FCS but including 0.6 µg/mL sonic hedgehog (SHH) (CGM-SHH) and equilibrate it to 37 °C when needed.
   4. Coat 6-well cell culture plates with 100 µg/mL poly-D-lysine for 1-2 h at RT for glia-cell removal. Afterwards wash twice with sterile ddH₂O and let the plates dry. Store the plates for maximum one week at 4 °C.
   5. For cultivation of CGNPs, coat 6-well cell culture plates with poly-L-ornithine (0.1 mg/mL) at 4 °C overnight. Afterwards, wash twice with H₂O for cell culture and let the plates dry.
      NOTE: The plates can be stored for a maximum of one week at 4 °C.
   6. Prepare 0.025 % trypsin (w/v) in HBSS/Glucose and equilibrate it to 37 °C when needed.
2. Neural Progenitor Isolation of Brain Tissue
   1. Isolation and optional cultivation of CGNPs (cerebellar granular neuron progenitors)
      1. Euthanize P5-7 animals by decapitation using scissors. Remove the scalp skin, open the skull, and remove the brain using small scissors and forceps. Isolate the cerebellum and transfer it to ice-cold HBSS/Glucose. Remove all meninges and blood vessels and transfer the cerebella into 15 mL tubes filled with cold HBSS/Glucose.
      2. Wash the cerebella three times with 10 mL ice-cold HBSS/Glucose (collect them by centrifugation at 650 x g for 5 min at 4 °C) and homogenize cerebella subsequently by gently pipetting up and down with a 1 mL pipette (maximum 2-3 times) to get 0.5-1 mm³ fragments.
      3. Add 5 mL of 0.025% trypsin in HBSS/Glucose and incubate the tissue under constant stirring in a 37 °C water bath for 15 min. Stop the digestion by adding 5 mL of CGM and collect the tissue by centrifugation at 650 x g for 5 min at 4 °C.
      4. Remove the supernatant and triturate the tissue with a 1 mL pipet tip in 1 mL CGM and transfer it to a new 15 mL tube.
         NOTE: It is important to avoid air bubbles.
         1. Add 5 mL CGM and incubate the mixture for 2 min on ice to settle tissue remnants. Transfer the supernatant into a new 15 mL tube. Add 2 mL CGM to the residual tissue and repeat the trituration procedure.
      5. Pool the supernatants (without tissue remnants, about 10 mL in total) and centrifuge at 650 x g for 5 min at 4 °C to collect the cerebellar cells. Resuspend the pellet in 10 mL CGM.
      6. Since astrocytes adhere faster and stronger to poly-D-lysine than CGNPs, plate the cells on 100 µg/mL poly-D-lysine coated 6-well-plates (up to 4 mL per well) and incubate for 20 min at 37 °C to remove the astrocytes.
      7. Shake the plate and collect the supernatant. Then centrifuge at 650 x g for 5 min at 4 °C in a 15 mL tube. Resuspend the pellet in 10 mL CGM and count the cells with a Neubauer counting chamber.
         NOTE: CGNPs are round, small, and show a halo when imaged using phase contrast microscopy.
      8. If required, seed the cells in 37 °C pre-warmed CGM on poly-L-ornithine-coated plates (3 x 10⁶ cells per 6-well) and incubate them at 37 °C, 5% CO₂ and 100% relative humidity.
         NOTE: Leaving CGM on the plates (and with that FBS) will result in the differentiation of the CGNPs into cerebellar granular neurons (CGNs).