I have done DRG cell culture (isolated from young rats) for patch clamp work. If you can send me the protocol you have used, I can give some pointers. You may have to use some coating on your plating surface like basement membrane or laminin. I may be reached at sudas_1 at yahoo dot com.

I have done DRG cell culture (isolated from young rats) for patch clamp work. If you can send me the protocol you have used, I can give some pointers. You may have to use some coating on your plating surface like basement membrane or laminin. I may be reached at sudas_1 at yahoo dot com.

Check the affinity for the substrate. You can try coating wells with either poly l lysine or pei. Good luck

Sorry i´ve forgot mention that i added Poly-L-Lysine into the wells, but it didn´t work. One moth before my last experiment with DRG cells, I added matrigel into the well, but i can´t see any differences in the morphology. Thanks so much for your comments!!!!! Actually, i am crazy because of that. I don´t know what i have to do.

You can follow the following method from our publication,

http://ajpcell.physiology.org/content/285/1/C64.long

Molecular cloning and characterization of a rat sensory nerve Ca2+-sensing receptor

DRG culture

DRG were isolated under aseptic conditions, trimmed of axons, and dispersed using a collagenase-based method as described by Supowit et al. (35). The dispersed cells were initially seeded on polyornithine-coated coverslips in F-12 medium supplemented with 10% heat-inactivated horse serum. After 48 h, the medium was replaced with serum-free F-12 enriched with N2 supplement (GIBCO).

http://ca3cx5qj7w.search.serialssolutions.com/OpenURL_local?sid=Entrez:PubMed&id=pmid:9519262

Alpha 2-adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons.

DRG neurons were prepared following a modified protocol initially described by Lindsay [20]. DRG (cervical, thoracic and lumbar; 40–45/animal) were dissected from male Sprague–Dawley rats (125–150 g) and collected in Ham's F-12 medium supplemented with 10% horse serum (growth medium). Ganglia were dissociated in 0.125% collagenase with a constant flow of 5% CO2/95% O2, washed, and then treated with 0.25% trypsin. After another wash, the ganglia were transferred to growth medium containing DNase (80 μg/ml) and soybean trypsin inhibitor (100 μg/ml). Single cell suspensions were obtained by trituration of enzymatically softened ganglia. Following additional washes, the dissociated neurons were plated in six-well culture dishes coated with polyornithine and maintained in growth medium at 37°C in 5% CO2. After 24 h, the cells were placed in serum free conditions. The medium was Ham's F-12 supplemented with insulin (5 μg/ml), transferrin (100 μg/ml), progesterone (20 nM), selenium (30 nM) and putrescine (100 μM, N2 Supplement, Gibco-BRL). The yield of neurons was approximately 1.5 to 2.0×105 from 40–45 ganglia. For these studies the dissociated DRG cells were plated at a density of 10,000 to 20,000 neurons/well. The cells were incubated at 37°C in a 5% CO2, 95% air atmosphere.

Hi Marian,

As mentioned the issue could be the substrate for cell attachment. However, if you are already using a proper coating with PDL or Matrigel it could also be an issue of the growth medium. If the cells attach and look good in the first 24 hours, but then later fail to grow and die off, this might be the most likely culprit. In particular you should pay close attention to the quality of the supplement for supporting neuron growth (either N2 or B27, etc) as these are know to have batch to batch variability with some batches providing inadequate support--particularly under less than optimal culture conditions. If you provide your culture medium recipe I am sure someone might be able to comment about how to optimize this, and the recipe by Chandra seems very reasonable for DRG culture.

Hi Jonathan, Thanks for your advice. But I’m in Cuba, so i use a brazilian´s protocol with some adaptations for our lab conditions. The only growth medium that we have now is DMEM (Sigma), supplement with 10 % bovine fetal serum (Sigma) and 5 % antibiotic. I was searching for months what reagent i could use for this culture, since our brazilian collegues give us this protocols, which use HBSS and DMEM medium. Lockly we had these reagents in our lab!

If you think that our growth medium is wrong, and perhaps that is what we have had problems, i will search for the others reagents which Chandra suggested me.

I appreciated your comments; it gives me another idea for my problem. Sorry I’m a beginner in cell culture, so i don´t have the tools to find, in the good way, the solution for the problems. Thanks again!!

Hi Marian, here in attach detailed protocol for DRG isolation, it's fully described step by step and possible problems issue,

regards,

Thanks!!! I really appreciate it. I´m very greatfull!!!

Hi Marian,

I do think the growth medium is likely the problem based on the fact you are only using DMEM with 10% FBS (generally only allows survival of glia). You need more support than just the factors in FBS for robust growth of most neurons in culture. I would recommend supplementing insulin and transferrin in particular (similar to Chandra's recipe above), as these are the key components of N2 supplement and are part of the complex formulation of the widely used B27 supplement. You should be able to get these from Sigma. Lastly, you should double check the amount of antibiotic as 5% doesn't seem right (too much, this would be 5g/100mL media!!). For example, pen/strep antibiotic is usually sold as a liquid 100X stock (5000U/mL each) that is diluted to 1X (50U/mL each) in the neuronal growth medium. Too much antibiotic can definitely damage or kill neurons. If you are not sure you can try omitting the antibiotic in a couple tries to see if this helps. Many people including myself culture in the absence of antibiotic and instead work on improving sterile technique. I hope you solve it!

You may try to coat the plate with 0.5 mg/ml poly-D-lysine overnight then washed with dd water for 6 times to completedly rinse the plate than plate with laminin at 20 ug/ml for 3-4 hours. Then just proior to plating, aspirate the laminin and (do not rinse with PBS or culture media) plate DRG cells suspension on the wells.

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 may try to coat the wells with Poly-D-Lysine overnight and rinse throughtly then continue to coat with laminin (354087, BD BioCoat, Lot: 3045819) for 3-4 hours. You may also add 10 ng/mL murine 2.5 S NGF into the mddium. Below is the poly-D info:

Poly-D-Lysine Cat# 3439-100-01

Lot# 21487J10

Tel: 1-800-873-8443

Supplier: Cultrex

Hope it helps!

Dear Dr.Chang-Ning Liu. Thanks. 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.What media combination do you recommend ?