Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies

Summary

[INSERT FIGURE1 HERE]

Figure 1: Spinal column dissected and spinal cord released from a 3 day-old neonatal mouse. The arrow in (a) points towards the caudal end of the spine, where a needle is inserted to release the spinal cord. A released spinal cord (b) is shown submerged in PBS.

[INSERT FIGURE2 HERE]

Figure 2: Density gradient, with cells added-on after centrifugation. The layers are outlined and better visualized on the cartoon depiction. The most superficial layer (0) is generally debris from the spinal cord tissue. Layer 1 is rich in oligodendrocytes and astrocytes. Layers 2 and 3 contain the majority of neurons. While layer 3 contains neurons with the highest purity, some supporting cells (i.e., astrocytes and oligodendrocytes) are found in layer 2. The pellet at the bottom mainly contains microglial cells.

Abstract

Source: Eldeiry, M. et al. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (2017)

In this video we describe a step-by-step protocol to isolate spinal cord neurons from a neonatal mouse for in vitro studies.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Harvesting Spinal Cords NOTE: All instruments should be autoclaved (135 °C and 30 psi for 4 x 7 min cycles) for sterility. Euthanize 1–3 day-old C57BL/6 mouse pups in a chamber with isoflurane. Wait 30 s after the cessation of movement and pinch the leg to confirm …

Representative Results

Figure 1: Spinal column dissected and spinal cord released from a 3 day-old neonatal mouse. The arrow in (a) points towards the caudal end of the spine, where a needle is inserted to release the spinal cord. A released spinal cord (b) is shown submerged in PBS. <img alt="Figure 1" src="…

Materials

Hibernate A Medium – 500 mL	Thermo-Fisher	A1247501	https://www.thermofisher.com/ order/catalog/product/A1247501
Hibernate A Minus Calcium – 500 mL	Brainbits	HA-Ca	http://www.brainbitsllc.com/ hibernate-a-minus-calcium/
Glutamax 100X – 100 mL	Thermo-Fisher	35050061	https://www.thermofisher.com/ order/catalog/product/35050079
B27 Supplement 50X – 10 mL	Thermo-Fisher	17504044	https://www.thermofisher.com/ order/catalog/product/17504044
Papain, Lyophilized – 100 mg	Worthington	LS003119	http://www.worthington- biochem.com/pap/cat.html
Neurobasal A Medium – 500 mL	Thermo-Fisher	10888022	https://www.thermofisher.com/ order/catalog/product/10888022
Penicillin-Streptomycin (10,000 U/ mL)	Thermo-Fisher	15140122	https://www.thermofisher.com/ order/catalog/product/15140122
Poly-D-Lysine (PDL) hydrobromide- 5 mg	Sigma-Aldrich	P6407-5MG	http://www.sigmaaldrich.com/ catalog/product/sigma/p6407? lang=en&region=US
Mouse Laminin – 1 mg	Thermo-Fisher	23017015	https://www.thermofisher.com/ order/catalog/product/23017015
Trypan Blue – 20 mL	Sigma-Aldrich	T8154-20ML	http://www.sigmaaldrich.com/ catalog/product/sigma/t8154? lang=en&region=US
OptiPrep Density Gradient Medium-250 mL	Sigma-Aldrich	D1556-250ML	http://www.sigmaaldrich.com/ catalog/product/sigma/d1556? lang=en&region=US
Dichlorodimethylsilane (DMDCS, Sigma Silicoat)	Sigma-Aldrich	440272-100ML	http://www.sigmaaldrich.com/ catalog/product/aldrich/440272? lang=en&region=US
Chloroform	Sigma-Aldrich	288306-1L	http://www.sigmaaldrich.com/ catalog/product/sial/288306? lang=en&region=US
Glass Pippette – 9"	Sigma-Aldrich	13-678-20C	http://www.sigmaaldrich.com/ catalog/product/sigma/cls7095d9? lang=en&region=US
Pipette bulb – 5 mL	Sigma-Aldrich	Z186678-3EA	http://www.sigmaaldrich.com/ catalog/product/aldrich/z186678? lang=en&region=US&cm_sp=Insite- _-prodRecCold_xviews-_- prodRecCold10-1
BRAND® Petri dish, glass – 60 x 15 mm	Sigma-Aldrich	BR455717-10EA	http://www.sigmaaldrich.com/ catalog/product/aldrich/br455717? lang=en&region=US
Sterile 24-Well Cell Culture Plate	Sigma-Aldrich	M8812-100EA	http://www.sigmaaldrich.com/ catalog/product/sigma/m8812? lang=en&region=US