I tried to culture DRG neuron with Neurobasal A, B27, L-Glutamine P/S and 50ng/ml NGF for electrophysiology purpose. My cells do not look healthy. I would appreciate if you input your protocol. Thanks.
You can refer to Mahendra singh (Univ of Navada, Reno) on Researchgate. He is very experienced in electrophysiology and calcium imaging of TRPV1 in DRG cultures.
Hi Saikat,
I've been working on TRPV1 signals in DRGs and vagal sensory neurons. I use the complete F12 medium and the cells thrive in it. I get processes after 16h without any growth factors and the cells are fully responding to capsaicin after a week.
here are the details of the media:
Life Technologies (Gibco) Ham's F-12 Nutrient Mixture (F-12) Ref 21765-029 (contains L-glutamine and Phenol Red)
Supplement it with 10% heat inactivated FBS and 1% penicillin/streptomycin
They are cultured in 5% CO2.
I do not add any NGF ! that messes up the phenotype of the cells badly as 48h after isolation if NGF is present I don't get the same responses to TRP agonists when compared to cell incubated without it (all from the same tissue).
I used Medium M199 (125ml) + Glucose (0.625g) + Penicillin/Streptomycin (1.25 ml) + mNGF (50ng/ml) + 10% FBS. The cultured DRG cells responded to nicotine as well as ATP. I recorded the responses with calcium imaging and voltage-sensitive dye imaging. But I never looked at TRP channels in the culture.
wouldn't omitting NGF prevent the proper growth of DRGs? or is this only true for embryonic DRGs?
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