In my experience, trypsin is too hard on neurons and destroys the axons. I am also afraid that centrifugation, and pipetting cause damage. Does anyone have experience in dissociating cultured neurons and can recommend the best method?
Dear Tatyana,
No matters how you do it, the detachment of primary neuronal cultures will cause a lot of stress to the neurons, so don't feel saad if some of these folks dye during the process, C'est la vie. That's why this procedure is also not recommended if you plan to study phenomenon arising shortly after re-plating like fast neurite outgrowth and neuron polarization. However, there are some tips to may try in order to diminish cell damage and increase survival. Try reducing the concentration of the substrate you use to attach the cells. For instance, if you use PLL or PDL you can use 10-50 ug/ml of it and neurons will still be happy and detachment will go easier. Also try reducing the concentration of trypsin to 0.025-0,05% and increase the incubation time. An alternative is using Papain, which is way more gentle than trypsin. For Papain, using the concentration recommended by the supplier, you will have to play around to find the best incubation time.
Best,
Hello Tatyana.
If cells are from embryonic or neonatal, they tend to make islands with long axons with neural stem cells aggregated. I use 1/10~1/5 dilution of a routine working concentration of trypsin-EDTA sol with patience. Although the axons are retreating upon the treatment, it doesn't mean that they are dying. I don't know what is your experiment, isolated cells are still active for further culture for ICC of other purposes. It is critical to make single cell suspension to avoid cell loss or aggregating together.
Probably, you need to adjust the initial cell concentration much lower, and single cell suspension before plating your cells. This would make the following procedures easier and safer.
Regards.
Have you tried Accutase? It tends to be more gentle for neuronal dissociation.
Thanks a lot for the answers. There are definitely some very helpful suggestions. Meanwhile, I've compared different methods that were mentioned here, and it seems that with Accutase (1:5 dilutions in 37C) I have more survival (my neurons are iPSC derived cortical neurons that tend to clump). I did not try papain, but this is definitely something worth considering.
Two pre-step issues that might help, in addition to the Accutase suggestions). 1). Gently wash the neurons with warmed divalent cation free PBS once (or twice) depending on cell line and density. 2). Apply cell grade EDTA for several minutes (5 -10 minutes). There are some cell lines that will lift with just cell grad EDTA. TrypLES is also slightly less traumatic, and cheaper than Accutase was at one time. Also there are other methods like citric saline (again, divalent free being better).
Hi - I use accutase. And my cells are the same: IPS-derived cortical neurons. My protocol: Rinse cells with PBS. Add accutase, incubate at 37oC for 8-10 minutes. Add medium to neutralize accutase. Collect cells by gentle scraping, triturate with 1 ml tip for a few times gently. Spin down the cells, re-suspend in N2B27 containing Neural culture medium (add growth factors as needed) and plate the cells. I usually see getting attached within an hour but will see extended neurites only after an overnight incubation.
Sravan, Since there is EDTA in the Accutase [ 0.5 mM EDTA•4Na ] using divalent cation free PBS should work better, for what it is worth.
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