- Home
- Protocols
-
Generation of Neuron-enriched Cultures (Method 1)
Published: Nov 5, 2011 DOI: 10.21769/BioProtoc.151 Views: 11021
Download PDF
Ask a question
Abstract
This protocol will guide you through the process for generating midbrain neuronal cultures from late embryo mouse brain. These cultures serve as a useful tool to study molecular pathways during neuronal death in various neurological disorders. This method uses cytosine β-D-arabinofuranoside in the cultures to suppress the proliferation of glial cells. Seven days after the seeding, the neuron-enriched cultures prepared following this protocol will contain less than 10% glia cultures. The concentrations of β-D-arabinofuranoside should be adjusted according to your culture condition. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.
Materials and Reagents
- Poly-D-lysine (Sigma-Aldrich, catalog number: P7280 )
- MEM (Life Technologies, Gibco®, catalog number: 11090-08 )
- D-Glucose
- Sterile PBS
- Trypan blue dye
- Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
- Heat-inactivated horse serum (HS) (Life Technologies, Gibco®, catalog number: 26050-088 )
- Non-essential amino acids (100 ml) (Life Technologies, Gibco®, catalog number: 11140-050 )
- Sodium pyruvate (100 ml) (Sigma-Aldrich, catalog number: S8636 )
- 200 mM L-glutamine (100 ml) (Life Technologies, Gibco®, catalog number: 25030-081 )
- Penicillin/streptomycin (100 ml) (Sigma-Aldrich, catalog number: P0781 )
- Neurobasal medium (Life Technologies, InvitrogenTM, catalog number: 12348-017 )
- 50x B27 serum-free supplement (10 ml) (Life Technologies, InvitrogenTM/ Gibco®, catalog number: 21103-049 )
- Poly-D-lysine solution (see Recipes)
- Maintenance culture medium (see Recipes)
- Treatment medium (see Recipes)
Equipment
- Cell culture incubator
- Centrifuges
- Dissection microscope
- Scissors and forceps
- Laminar hood
- 24-well plates
- 50 ml tube
- 10-ml pipet
Procedure
Category
Neuroscience > Cellular mechanisms > Cell isolation and culture
Cell Biology > Tissue analysis > Tissue isolation
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Post a Question
