Hallo everybody,
Could you please, help me? I am working with dorsal root ganglion neurons and I used many methods to have pure neurons but I doesnot work with me. firstly, I used antimitotic chemicals such as 1 µM Ara C alone for 2 weeks and percentage of non neuronal cells is very high . ALso, I used mix from (20µM FUdR +0.5µM Ara C) for one time treatment for DRGs culture of cells according to literature and after 7 days , non neuronal cells including schwann cells and fibroblast cells grow and they are not die. PLease, help me, If anybody worked with these neurons and know that How to stop growth of non neuronal cells. I attached some photos for cells after 8 days of treatment with mitotic inhibitors. Thanks in advance.
Dear Amira Elfarnawany,
As reported by Shen, et al. (2019), the timing of antimitotic application (after DRG neurons had been cultured for 48 h), at an optimal concentration, is critical to reduce non-neuronal cell growth while avoiding a significant reduction of neuronal yield.
https://journals.sagepub.com/doi/full/10.1177/0300060519855585
Additionally, other methods of purification may also be applied.
I hope this is helpful.
Good Luck
I do wonder if it is your cell extraction method rather than anti-mitotic treatment that is causing the issue. I followed the protocol attached (for mice) and had very few non-neuronal cells. Key step may be the density gradient centrifuge.
Dear Amira Elfarnawany,
The methods annotated by previous answers will function, however as far as I understand there is no way to obtain purely DRG neurons. There is always be some non-neuronal cells.
Enrique
Dear Amjad Hasan Bazzari,
Thanks so much for your answer. I will apply your advices in the next time of preperation of neurons. The problem may be in the time of applying antimitotic chemicals as I applied after seeding neurons for 24 hours. I will trz this time after 48hrs. Thanks again for your help.
Dear Steven Fagan ,
Thanks a lot for your answer. I donot know exactly the problem in way of extraction or antimitotic chemicals treatment as I used this protocol which reported by (Malin, et al.2007)for extraction of cells and it worked good with me from the first time but I think the problem in the way of purification of neurons from non neuronal cells after preperation.According to the protocol that you attached , I will use the 15% BSA purification for debris and meylin .Hopefully, it works this time as I used before, 3.5% BSA purification according to (Park et al .2021) and it didnot work with me. Also ,the problem may be as I used low conc of (UfdU) 20µM as final conc so I will try with 40µM in the next trial and I hope that the number of non neuronal cells decreased. Thanks so much for help .
Dear .Enrique Soto ,
Thanks so much for your answer. I agree with you that I cannot obtain 100% pure neurons but in my case, I noticed that the non neuronal cells including schwann cells and fibroblast cells grow very quickly and there is no effect for antimitotic inhibitors on my cells as compared with the results of other people who used the same concs of antimitotic chemicals . Till now , I donot know why it didnot work. I will try again with purification method which people mentioned in previous answers. Thanks again
Dear Amira Elfarnawany ,
I can recommend using freshly prepared Mitomycin-C (1 µg/mL, M4287, Sigma-Aldrich) for 2 hours. I used it to reduce the population of non-neuronal cells (primarily fibroblasts) when culturing iPSC-derived sensory neurons. Let me know if you have any questions regarding this.
/Christina
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