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

Summary

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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.

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.

1. 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 .......