概要

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

Published: April 30, 2023

概要

[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="…

開示

The authors have nothing to disclose.

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

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記事を引用
Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies. J. Vis. Exp. (Pending Publication), e20698, doi: (2023).

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