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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 &#176;C and 30 psi for 4 x 7 min cycles) for sterility.
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	<li>Euthanize 1&#8211;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 ...

spinal cord neuron isolation: a density gradient procedure to isolate neonatal spinal cord neurons for in vitro studies

Source: Eldeiry, M. et al. <a href="https://www.jove.com/video/55856" target="_blank">Spinal Cord Neurons Isolation and Culture from Neonatal Mice.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/20698/20698_Figure1.jpg" /> 
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="...

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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.

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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.

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

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 ...

To isolate spinal cord neurons, take a vertebral column of a neonatal mouse and place it onto a Petri dish. Flush the vertebral column with saline buffer to release the spinal cord. Transfer the spinal cord into cold neuronal medium to promote cell survival.
Next, mince the spinal cord and transfer the fragments into a tube containing fresh neuronal&#160; medium. Incubate the tube in a shaker water bath for acclimatization. After incubation, remove the spent medium and add the digestion medium. Digestive enzymes cleave the tissue matrix to loosen up the cells.
Perform trituration by aspirating the tissue fragments into the pipette and swiftly empty the components, releasing individual cells into suspension. Then, transfer this suspension into a tube containing density gradient medium and centrifuge.
Following centrifugation, the tube&#39;s superficial layer contains tissue debris. The first layer includes oligodendrocytes and astrocytes. The second and third layers comprise neurons, with the third layer having the highest purity. Lastly, the pellet contains microglial cells.
Collect the third layer into a fresh tube and add neuronal medium to dilute out the density gradient medium. Centrifuge to pelletize the neurons. Discard the supernatant and resuspend the pellet in a neurobasal medium to preserve the neurons for further experiments.

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

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