Hello, guys! I have been working on the primary oligodendrocyte precursor cells (OPCs) culture these days. The cells were from the cerebral cortices of 1-2 d-old C57BL/6 mice. On the 8th day of mixed glial cells culture, OPCs were collected and proliferated in DMEM/F12 + 20 ng/ml PDGF +20 ng/ml FGF +1% B27 for 4-5 days. Then the medium was switched to differentiation medium of DMEM/F12 + 30 ng/ml T3 (sigma: T6397)+ 20 ng/ml CNTF +1% B27. The differentiation period was about 7 days. Eventually I get the cells in the phase control picture below.
And here comes my questions:
1. The cells seem to be branched, but I don't think they are branched enough to form a net which should be like a spider net (I also provide a phase control figure of mature oligodendrocyte from published paper of Najm FJ below). So what is the problem with my cells? What should I do? To wait longer? But 7 days seem to be enough for differentiation. Or in my phase control figure, the cells are still OPCs which haven't differentiate yet?
2. What's more, I have stained them with GFAP and MBP. The cells can be stained with both GFAP and MBP! So I am totally confused! I want to ask that can OPCs differentiate into astrocytes even under a circumstance free of BSA???
3. I have got a MBP staining photo from abcam. And the cells provided there were claimed to be MBP-positive oligodendrocytes which has the same morphology of mine. So can I presume that I have differentiate OPCs into mature oligodendrocytes successfully?
Thank you for your help!!
I came across this paper before: https://www.ncbi.nlm.nih.gov/pubmed/18045844 saying that NG2+ cells can differentiate both into oligodendrocytes and protoplasmic astrocytes. Did you stain your primary OPC to access the purity? Did you coat your culture vessel?
Dear Yi Xie,
we work with human oligodendrocytes so antibodies may be different, but here is a list of antibodies we use and work very well for olidondendrocytes.
MBP/ SMI99 Mouse Monoclonal COVANCE 1:1000
NOGO-A Rabbit Polyclonal Santa Cruz 1:500
OLIG1 Mouse Monoclonal Millipore 1:200
Moreover, in our protocol we need 6 weeks to differnetiate the oligodendrocytes. Not sure if you will need so much in mice... but i human we definitelly need a lot of time.
Best regards,
David Pamies
Dear Yi,
Based on cell morphology of your culture, they are mature oligodendrocytes. MBP is a good mark for mature cells, but you can also use others such as O4. In the serum free culture condition, some astrocytes also behave multi-processed morphology, but they should not MBP or O4 positive. Good luck!
Deal Katie,
Thanks for your answer! I have stained my OPCs with NG2 and the majority of cells can be stained positive. And yes, I have coated my culture vessel and dish with PLL.
Dear David,
I am afraid I cannot induce OPCs differentiation for that long time. Thank you very much for the recommended antibodies! I will try~
Dear James,
Thanks a lot! You make me feel relieved~I have stained the cells with MBP (ab40390) and oligodendrocyte specific protein (ab53041). However, the cells can be stained positive which are the brached cells but not the highly-processed ones.
I have attached another phase control figure of my OPCs before differentiation. A PI in my lab told me that they are indeed OPCs in good conditions. After these days trying, I have changed the recipe of my differentiation medium according to the paper: https://www.ncbi.nlm.nih.gov/pubmed/25366898 and https://www.ncbi.nlm.nih.gov/pubmed/17546009. Thus now the differentiation medium is: DMEM/F12 + 400 ng/ml T3 + 20 ng/ml CNTF + 1× NAC + 0.4% BSA + 0.1% ITSS. Even some OPCs die when the differentiation medium is added, those survived can differentiate very well and lots of processed mature oligodendrocytes can be observed now. And I will keep trying and keep you all updated!
I really appreciate all your suggestions!!! A Million Thanks!!!
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