Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons

Abstract

Source: Er, S., et al. Studying Pre-formed Fibril Induced α-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons. J. Vis. Exp. (2020).

The video demonstrates the process of isolating and culturing dopamine neurons from the midbrain of mouse embryos. The isolated ventral midbrain floors are dissociated using enzymes. The resulting dopamine neurons are harvested and transferred into micro-islands inside multi-well plates. The cells are incubated with a dopamine neuron medium to maintain the cells at a high density inside the micro-islands.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation

1. Prepare dopamine neuron medium (DPM) with 0.46% D-glucose, 1% L-glutamine, 1% N2, 0.2% primocin, completed with Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12). Filter the DPM after mixing the ingredients. Store DPM at 4 °C and warm each aliquot only once.
   NOTE: DPM should not contain Glial cell line-derived growth factor (GDNF), as it will reduce α-synuclein accumulation in dopamine neurons.
2. Prepare siliconized glass pipet....

Results

Figure 1: Dissection of midbrain floor from E13.5 mouse embryo. (A) Cutting locations at the hindquarter of the head is marked with black arrows and white dashed lines. (B) The piece was removed from the rest of the embryo. The re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.4% Trypan Blue stain	Gibco, Thermo Scientific, Waltham, Massachusetts, USA	15250061
15 ml CELLSTAR Polypropylene tube	Greiner Bio-One GmbH, Frickenhausen, Germany	188261
4',6-diamidino-2-phenylindole (DAPI)	Sigma-Aldrich, St. Louis, Missouri, USA	10236276001
5 M HCl	N/A	N/A	Media kitchen, Institute of Biotechnology, University of Helsinki
5 M NaOH	N/A	N/A	Media kitchen, Institute of Biotechnology, University of Helsinki
96-well ViewPlate (Black)	PerkinElmer, Waltham, Massachusetts, USA	6005182
99.5% Ethanol (EtOH)	Altia Oyj, Rajamäki, Finland	N/A
AquaSil Siliconizing Fluid	Thermo Scientific, Waltham, Massachusetts, USA	TS-42799
Autoclaved 1.5 ml microcentrifuge tube	N/A	N/A	Media kitchen, Institute of Biotechnology, University of Helsinki
Bioruptor sonication device	Diagenode, Liege, Belgium	B01020001
Ca2+, Mg2+ free Hank's Balanced Salt Solution (HBSS)	Gibco, Thermo Scientific, Waltham, Massachusetts, USA	14175-053
CellProfiler and CellAnalyst software packages	N/A	N/A	free open-source software
Centrifuge 5702	Eppendorf AG, Hamburg, Germany	5702000019
CO2 Incubator	HeraCell, Thermo Scientific, Waltham, Massachusetts, USA	N/A
Counting chamber	BioRad Inc., Hercules, California, USA	1450015
DeltaVision Ultra High Resolution Microscope with air table and cabinet	GE Healthcare Life Sciences, Boston, Massachusetts, USA	29254706
Deoxyribonuclease-I (Dnase I)	Roche, Basel, Switzerland	22098700
DMEM/F12	Gibco, Thermo Scientific, Waltham, Massachusetts, USA	21331–020
Dulbecco's buffer	N/A	N/A	Media kitchen, Institute of Biotechnology, University of Helsinki
Fetal Bovine Serum (FBS)	Gibco, Thermo Scientific, Waltham, Massachusetts, USA	10500056
D-glucose	Sigma-Aldrich, St. Louis, Missouri, USA	G8769
Phospohate Buffer Saline (PBS)	N/A	N/A	Media kitchen, Institute of Biotechnology, University of Helsinki
Primocin	InvivoGen, San Diego, California, USA	ant-pm-1, ant-pm-2
poly-L-ornithine	Sigma-Aldrich, St. Louis, Missouri, USA	P4957
Triton X-100	Sigma-Aldrich, St. Louis, Missouri, USA	11332481001
trypsin	MP Biomedicals, Valiant Co., Yantai, Shandong, China	2199700