Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

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 ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B27 Supplement (50x)	Life Technologies	17504044	Category: Cell culture Abbreviation/Comment: For CCM
DMEM-F12	Life Technologies	11320-033	Category: Cell culture Abbreviation/Comment: For CGM
Fetal Bovine Serum 10% (v/v)	Gibco	10082147	Category: Cell culture Abbreviation/Comment: For CPC isolation and CGM
Glucose	Sigma-Aldrich	G5767	Category: Cell culture Abbreviation/Comment: For CGNP isolation
Glutathione (1.25 mg/ml)	Sigma-Aldrich	G4251	Category: Cell culture Abbreviation/Comment: For CCM
Glycine	Carl Roth	3187	Category: ChIP Abbreviation/Comment: For cell fixation
Hank's Balanced Salt Solution	Life Technologies	14025-100	Category: Cell culture Abbreviation/Comment: HBSS
L-glutamine (200 mM)	Life Technologies	25030081	Category: Cell culture Abbreviation/Comment: For CCM
Laminin	Sigma-Aldrich	L2020	Category: Cell culture Abbreviation/Comment: For CPC culturing
N2 supplement	Life Technologies	17502048	Category: Cell culture Abbreviation/Comment: For CGM
Neurobasal medium	Gibco	21103049	Category: Cell culture Abbreviation/Comment: For CCM
Penicillin-Streptomycin-Neomycin 1% (v/v)	Life Technologies	15640055	Category: Cell culture Abbreviation/Comment: PSN, for CCM and CGM
Phosphate buffered saline	Life Technologies	10010023	Category: Cell culture Abbreviation/Comment: PBS, for CPC isolation
Poly-D-lysine	Sigma-Aldrich	P6407	Category: Cell culture Abbreviation/Comment: For CGNP isolation
Poly-L-ornithine hydrobromide	Sigma Aldrich	P3655	Category: Cell culture Abbreviation/Comment: For CPC culturing
Potassium chloride	Thermo Fisher	AM9640G	Category: Cell culture Abbreviation/Comment: KCl, for CGM
Sonic hedgehock (SHH)	Sigma-Aldrich	SRP6004	Category: Cell culture Abbreviation/Comment: For CGNP isolation
Superoxide dismutase (1mg/ml)	Sigma-Aldrich	S7571	Category: Cell culture Abbreviation/Comment: For CCM
Trypsin-EDTA 0,05% (w/v)	Sigma Aldrich	59417C	Category: Cell culture Abbreviation/Comment: For CPC isolation