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

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  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'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.