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Method Article

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

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July 8th, 2025

In This Article

Abstract

Source: Bovio, P., et al., Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark. J. Vis. Exp. (2018)

This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup's cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

Protocol

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).

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Disclosures

No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
B27 Supplement (50x)Life Technologies17504044Category: Cell culture
Abbreviation/Comment: For CCM
DMEM-F12Life Technologies11320-033Category: Cell culture
Abbreviation/Comment: For CGM
Fetal Bovine Serum 10% (v/v)Gibco10082147Category: Cell culture
Abbreviation/Comment: For CPC isolation and CGM
GlucoseSigma-AldrichG5767Category: Cell culture
Abbreviation/Comment: For CGNP isolation
Glutathione (1.25 mg/ml)Sigma-AldrichG4251Category: Cell culture
Abbreviation/Comment: For CCM
GlycineCarl Roth3187Category: ChIP
Abbreviation/Comment: For cell fixation
Hank's Balanced Salt SolutionLife Technologies14025-100Category: Cell culture
Abbreviation/Comment: HBSS
L-glutamine (200 mM)Life Technologies25030081Category: Cell culture
Abbreviation/Comment: For CCM
LamininSigma-AldrichL2020Category: Cell culture
Abbreviation/Comment: For CPC culturing
N2 supplementLife Technologies17502048Category: Cell culture
Abbreviation/Comment: For CGM
Neurobasal mediumGibco21103049Category: Cell culture
Abbreviation/Comment: For CCM
Penicillin-Streptomycin-Neomycin 1% (v/v)Life Technologies15640055Category: Cell culture
Abbreviation/Comment: PSN, for CCM and CGM
Phosphate buffered salineLife Technologies10010023Category: Cell culture
Abbreviation/Comment: PBS, for CPC isolation
Poly-D-lysineSigma-AldrichP6407Category: Cell culture
Abbreviation/Comment: For CGNP isolation
Poly-L-ornithine hydrobromideSigma AldrichP3655Category: Cell culture
Abbreviation/Comment: For CPC culturing
Potassium chlorideThermo FisherAM9640GCategory: Cell culture
Abbreviation/Comment: KCl, for CGM
Sonic hedgehock (SHH)Sigma-AldrichSRP6004Category: Cell culture
Abbreviation/Comment: For CGNP isolation
Superoxide dismutase (1mg/ml)Sigma-AldrichS7571Category: Cell culture
Abbreviation/Comment: For CCM
Trypsin-EDTA 0,05% (w/v)Sigma Aldrich59417CCategory: Cell culture
Abbreviation/Comment: For CPC isolation

Tags

Cerebellar Granule Neuron ProgenitorsDifferential AdhesionPoly D Lysine CoatingPoly L Ornithine CoatingTrypsin DigestionCell Culture MediaCentrifugationNeubauer Counting ChamberAstrocyte RemovalProgenitor Cell Differentiation