Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

Abstract

Source: Weinert, M. et al., Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains. J. Vis. Exp. (2015)

This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dissociation of Ventral Midbrain Cells

1. Transfer the pieces of the ventral midbrain under a laminar flow hood. Remove the Hanks' balanced salt solution (HBSS) and add 1 ml pre-warmed (37 ºC) 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) into the conical tube (volumes enough for up to 12 pieces of ventral midbrain). Incubate the tissue at 37 ºC for 5-10 min.
2. Remove trypsin-EDTA under the hood and add 1 ml de-activation medium (the ser....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's modified Eagle medium nutrient mixture F-12	Invitrogen	11330
Hanks' Balanced Salt Solution (HBSS) (1X), liquid	Invitrogen	24020-117
Fetal bovine serum, heatinactivated (FBS)	Invitrogen	16140
N2 Supplement (100X), liquid	Invitrogen	17502-048
D-(+)-Glucose solution (45% (wt/ vol) in water	Sigma	G8769
Bovine serum albumin BSA	Sigma	A9430
Laminin from Engelbreth-HolmSwarm murine sarcoma basement membrane	Sigma	L2020
Penicillin/streptomycin	Invitrogen	15070
Trypsin (0.05% (wt/vol)	Invitrogen	25300
Bovine serum albumin (BSA) cell culture tested	Sigma	A9418
Phosphate-buffered saline (PBS)	Sigma	P3813
Poly-L-ornithine, 0.01% solution	Sigma	P4957
Trypan blue solution (0.4% (wt/vol))	Biowhittaker	17-942E