Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells
成績單
To harvest the dorsal root ganglia, transfer the first embryo into a 10-centimeter culture dish containing room-temperature PBS under a dissecting microscope in the prone position and insert micro-dissecting forceps along either side of the spinal cord.
Using blunt dissection, separate the spinal cord from the surrounding soft tissue, using forceps to excise the spinal cord along the neck opening and tail stub. Remove the residual soft tissue from the freed spinal cord until only the spinal cord, nerve roots, and attached dorsal root ganglion remain.
Detach the individual dorsal root ganglia from their connecting nerve roots. Then, use a pipette pen equipped with a 1-milliliter pipette tip to transfer up to 100 dorsal root ganglia into a 1.5-milliliter microcentrifuge tube of PBS.
Collect the ganglia by centrifugation, and re-suspend the pellets in 200 microliters of recombinant enzymatic cell dissociation reagent per tube. After 10 minutes at 37 degrees Celsius, centrifuge the dorsal root ganglia again using a 200-microliter pipette tip to gently triturate the pellets in dorsal root postganglionic neuron maintenance medium.
Seed the dorsal root ganglion cells at 5 x 103 cells per square centimeter onto Poly-D-Lysine laminin-coated six-well plates in 1.5 milliliters of dorsal root ganglion neuron maintenance medium per well.
After two days in culture at 37 degrees Celsius and 5% CO2, wash the cells and return the neuronal cell cultures to the incubator in fresh dorsal root ganglion neuron purification medium for another two days.
After 3 to 4 maintenance and purification cycles, the dorsal root ganglion cell cultures should test positive for the neuronal marker Tuj-1 and negative for the glial cell marker S100 beta.
On day 7 of the Schwann cell-like cell culture, rinse the cells in PBS, followed by dissociation with 0.5 milliliters of recombinant enzymatic cell dissociation reagent per well at 37 degrees Celsius for 5 minutes. Resuspend the cell pellet in co-culture medium and seed the Schwann cell-like cells onto the purified dorsal root ganglion neuron culture at 1 x 103 cells per square centimeter for 14 days of co-culture at 37 degrees Celsius and 5% CO2.
