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.
We use 8-weeks old mice and the Malin protocol (https://pubmed.ncbi.nlm.nih.gov/17401349/) 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.
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.
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.