Adult DRG explants or dissociated cultures for axonal degeneration studies?

Does anyone have any experience using adult DRGs, either as explants or dissociated cultures, for axonal degeneration studies? My preference is to use adult because the process/disease I want to study occurs in adults, and also I’d like the full mature repertoire of non-neuronal cells like SGCs, macrophages, etc. to be present. I don’t know if the neuropathic process I want to study is a ‘neuron intrinsic’ one or if it requires other cells, so I don’t want to just have neurons all alone in a dish.

@adgaudet @liz @tberta @thicunha

Most all papers use embryonic DRGs. I’m assuming because they grow more readily and so you have more axon to quantify. But apart from that, any other reason that adult DRG shouldn’t work?

I did find a few recent papers using adult DRG:

  • They use adult DRG in Xona microfluid chambers and look at axon fragmentation


But, in contrast, this paper from Neto et al. suggests adult explants don’t grow much axon

Latremoliere et al. adapted the “spot culture” method that is normally used with embryonic DRG and got it going with adult DRG


We use 8-weeks old mice and the Malin protocol ( to prepare our cultures of dissociated DRG neurons. We obtain a good density of neurites (see image below, tubb3 staining, 3-4 days) in these cultures that we have successfully used to test neurodegeneration and potential neuroprotective drugs.

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Wow @tberta that’s really nice! Thanks for sharing with us. Are you counting neurites or axon length? Also are you using anti-mitotic agents? Unlike most axon degen people, I want the non-neuronal cells to be present, in case they are playing a role.

For the moment, we have just measured the neurite length. We did not use anti-mitotic agents, so I suspect that we have several non-neuronal cells in these cultures, too.

Dear @achamess. We have some ongoing projects and we have established the protocol for DRG explants similar to Good luck.

I am late to this thread, but in case anyone is still reading this, I have an update to our publication (the Malin protocol cited above): simply increasing the duration of the second enzyme incubation from 10 minutes to 20 minutes increases the yield of healthy isolated neurons, just do as little trituration as possible (we do 3-5 strokes each with a larger bore glass pipet followed by a smaller bore pipet, as in the protocol), too much trituration winds up killing a lot of your cells. For postnatal day 2 to adult dissociated neurons, you don’t need trophic factors for small neuron survival, but if you are trying to study large diameter neurons in vitro you will probably need to add NT3 and/or BDNF to keep them healthy.

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Thank you, @dmolliver. This is very helpful and still of current interest.

Thank you @dmolliver for sharing the updates on your widely used protocol. Thanks for joining Pain Researcher as well.

@dmolliver Do you have any thoughts on how best to count neurons/cells accurately for plating?

We’re doing some imaging assays where it’ll probably be important to consistently have the same number of cells on glass. What’s the best way to count? Hemocytometer and some live cell dye (calcein)?