All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Preparations
- Preparations for CGNP (cerebellar granular neuron progenitor) isolation
- Organize an appropriate mating of mice to generate P5-P7 animals to isolate the cerebellum (3-5 animals per ChIP and condition).
- Prepare HBSS/Glucose by adding 6 mg/mL glucose to the HBSS buffer.
- 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.
- 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.
- 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.
- Prepare 0.025 % trypsin (w/v) in HBSS/Glucose and equilibrate it to 37 °C when needed.
- Neural Progenitor Isolation of Brain Tissue
- Isolation and optional cultivation of CGNPs (cerebellar granular neuron progenitors)
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
