Hi!
Lately I have been trying to isolate and culture DRG from mice, but I am having some issue with it. I am following the ptocol of C. Sokol (2021); birefly:
- First, I coat the plate with a 30ul droplet of laminin 10 ug/ml.
- After picking the DRG, I digest them with Collagenase/DispaseII during 15 min at 37ºC.
- After washing DRG with DMEM, I resusend them in culture media supplemented with NGF and GDNF.
- I triture them with a syringe and different gauge needle (18G, 23G and 25G).
- Finally, I put then in culture.
I do not know if there is anything wrong in the protocol because after culturing them I am able to see DRG bodies, but any neural network after 24h in cultre.
Thank you!
Hi, I finally manage to isolate DRGs from mouse spinal cords. And I get enough DRG from one mouse (around 60,000 DRGs).
I don't know what you do not get enough neurons. Once I picked up the DRGs, I double-check de intervertebral spaces to make sure I got them all.
Hope this helps you!
I also picked up many DRGs. About 20-25 per mouse. I used 1.25mg
/ml collagen type I for 15 minutes to isolated.
However, few neuron was left after triturating.
Picture as follow:
Would you like to share a video of your digestion process FROM picking up the DRGs to plating cell? I will be honored to receive your guidance.
- For digesting DRGs I use a collagenaseA/disapseII mix for 15 min at 37ºC shaking and then I culture them in a laminine-coated plate; I follow this protocol (PMID 33615276).
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