Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Summary

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Abstract

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

Protocol

1. Poly-D-Lysine (PDL): Preparation

1. Add 5 ml of sterile ddH₂O to 5 mg of PDL to obtain a stock solution of 1 mg/ml.
2. Mix stock solution by pipetting several times.
3. Use immediately or store Poly-D-Lysine Solution at 2-8 °C.

2. Poly-D-Lysine (PDL): Coating Plastic Cell Culture Dishes

1. Dilute the PDL stock solution with sterile ddH₂O to the final concentration of 10 μg/ml.
2. Pipette enough solution into a 60 mm dish to cover culture surface area (3 ml for a 60 mm dish).
3. Rock gently to ensure even coating of the culture surface.
4. Incubate coated pl....

Discussion

The method of dissection and culture of rat hippocampal and cortical neurons described here allows performing experiments using nearly pure neuronal cultures grown in a chemically defined medium (Figure 3). Although protocols for culturing nearly pure neurons in serum-free media have been previously described^2,3,4, there are important changes made in our method. Different from traditional protocols (i.e. Banker et al.)⁵, we have replaced trypsin with TrypLE Express, a more gentle di.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neurobasal	98%
B27	2%
Glutamax	0.5 mM
Table I. Neurobasal/B27 complete medium.
Glucose	16 mM
Sucrose	22 mM
HEPES	10 mM
NaCl	160 mM
KCl	5 mM
Na2HPO4	1 mM
KH2PO2	0.22 mM
Gentamicin	50 μg/ml
Fungizone	250 ng/ml
pH	7.4
Osmolarity	320-330 mOsm
Table II. Dissection medium.
Neurobasal/B27 complete medium	240
Trypan Blue Stain 0.4%	250
Total	490
Table III. 50x Counting solution.
Hibernate E	Brainbits	767171
Neurobasal	GIBCO, by Life Technologies	21103-049
B27	GIBCO, by Life Technologies	17504-044
Fungizone	GIBCO, by Life Technologies	15290-018
Gentamicin sulfate	Sigma-Aldrich	G1264
Glutamax 200 mM	GIBCO, by Life Technologies	35050
TrypLE Express w/o phenol red	GIBCO, by Life Technologies	12604
Cytosine-β-D-arabinofuranoside hydrochloride	Sigma Aldrich	C6645
Poly-D-Lysine	Sigma Aldrich	P6407
Laminin 1 mg/ml	EMD Millipore	CC095
HEPES	Sigma-Aldrich	H3375
Trypan Blue Stain 0.4%	GIBCO, by Life Technologies	15250
Table IV. Specific reagents.
Stereo Microscope	Olympus Corporation	SZ61
Large Forceps	Fine Science Tools	11022-14
Fine-tipped forceps	Moria	MC40B
Micro fine-tipped forceps	Moria	MC31
Razor-sharp scissors	Roboz Surgical Instruments Co.	RS-6820
Micro Dissecting scissors	Fine Science Tools	91460-11
Micro Dissecting Curved scissors	Fine Science Tools	14067-11
Glass 2-chamber slides	Lab-Tek	154461
60 mm dishes	BD Biosciences	353002
100 mm dishes	Corning	430167
15 ml tubes	BD Biosciences	352099
1.5 ml cryo-tube vial	Nalge Nunc international	375353
Table V. Specific equipment.