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

To begin this procedure, separate the head from the body of the euthanized mouse pups using scissors. Next, stabilize the tail and arms on the procedure table with the dorsal side facing up. Then, cut the skin off using the curved iris scissors. After that, cut the spinal cord from the lumbar region just above the hips and then cut both sides of the thorax to separate it from the body.

Wash the samples sequentially in three 10-centimeter Petri dishes containing 5 milliliters of sterilized PBS for 10 seconds each to remove excess tissue. Subsequently, insert a 22-gauge needle and syringe filled with 5 milliliters of sterilized PBS into the caudal end of the spinal column and flush cranially, allowing the cord to exit into a fourth Petri dish.

Transfer the spinal cord in a 15-milliliter tube with 5 milliliters of HABG on ice. Avoid crushing the spinal cord and repeat the procedures for the rest of the pups. Ideally, this process should take no more than 30 minutes for one group of pups to ensure healthy neuron isolation.

To prepare the density gradient, prepare each of the four layers in four 15-milliliter tubes. Then, add 1 milliliter of each layer into a new 15-milliliter tube. Start with layer 1 at the bottom and add sequentially until reaching layer 4 at the top. Avoid disturbing the layers while adding and any vigorous movements that may cause mixing. Next, add the digestion media to the minced tissue. Place the sample in a shaker in the water bath at 30 degrees Celsius. Afterward, remove the tissue from the water bath and allow it to settle for a few minutes.

To perform trituration, aspirate the excess digestion medium. Suspend the tissue in 2 milliliters of HABG. Using a narrow-bore pipette, triturate 10 times in 45 seconds and avoid introducing air as it will significantly decrease the viable yield. Trituration is one of the most critical steps of the procedure. The tissue should be carefully drawn into and emptied from the fire-polished pipette, avoiding excess vigor, which can lead to neuronal death, or being too gentle, which will result in a low yield.

Following that, aspirate the top 2 milliliters of the supernatant and transfer it into a new 15-milliliter tube labeled "collection". Repeat the collection procedures two more times to yield 6 milliliters of supernatant. Next, slowly transfer the collection tube contents into the gradient tube prepared previously, avoiding the disruption of the gradient.

To purify the neurons, centrifuge the gradient tube for 15 minutes at 800 x g and 22 degrees Celsius. Collect the desired layers and transfer them in a new 15-milliliter tube. For the highest purity neuron isolation, collect layer 3. For more yield with less purity, collect layers 2 to 3.

Next, dilute the density gradient by adding 5 milliliters of HABG to the newly collected layers. Centrifuge it at 200 x g for 2 minutes at 22 degrees Celsius. After 2 minutes, discard the supernatant and resuspend the cells in 5 milliliters of HABG by flicking the pellet. Subsequently, centrifuge the sample at 200 x g for 2 minutes at 22 degrees Celsius again. Then, discard the supernatant and resuspend the cells in 3 milliliters of neurobasal medium by flicking the pellet.