Experiments on isolation of dorsal root ganglion of chicken embryo
Experiments on isolation of dorsal root ganglion of chicken embryo
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Isolation of dorsal root ganglia in chick embryos
Materials and Instruments
Enzyme solutions DRG Move 1. Prepare the enzyme solution in a 15 ml centrifuge tube before autopsy and place in a 37°C water bath. For more product details, please visit Aladdin Scientific website.
Centrifuge tubes Petri dishes Dissecting microscopes
2. Dissect the embryos and keep the tissues moist throughout the dissection.
(1) Kill the embryos, remove the legs and place them in 100 mm Petri dishes.
(2) Using blunt-tipped scissors, make an incision along the midline of the abdomen and roll the skin to the sides until the viscera can be removed.
(3) The body cavity was rinsed with CMF-PBS.
(4) Carefully remove any adherent tissue from the spine with fine forceps or by blowing with CMF-PBS.
3. Under a dissecting microscope, carefully tear the spinal cord from the spinal cord with fine forceps. Remove the sympathetic ganglia, the thin strips on either side of the spinal cord on the DRG.
4. Cut the ganglion from the nerve trunk with micro-spring scissors, remove the DRG from the spinal cord with fine forceps, and place in a 35 mm Petri dish containing 1 ml CMF-PBS.
5. Separate the appropriate amount of DRG, ensuring that there is no adherent tissue or nerve trunk, for a period of no more than 45 minutes.
6. Add 1 ml of CMF-PBS containing DRG to the pre-warmed enzyme solution and incubate for 8 or 13 minutes at 37°C in a water bath, inverting the tubes every 3 minutes.
7. Terminate the enzyme reaction by adding 1 ml of FBS and precipitate the DRG. wash the DRG with CMF-PBS and precipitate again.
8. Suspend DRG in 1 ml complete medium supplemented with NGF (37°C). Blow the cells gently (6 times) with a Pasteur pipette with the inner diameter of the tip burnt to half. If a tissue mass remains, allow it to settle and transfer the cell suspension to another 15 ml sterile centrifuge tube. Repeat the blowing away of residual tissue mass and mix the samples twice.
9. Count the dispersed cells and inoculate as needed at the appropriate density. 