I've been working with oligodendrocytes for a while, but I have trouble getting them to myelinate DRG axons. I based the experiment off of Jonah Chan and Ben Barres' culture (Chan et al., 2004).I start with a pure culture of dissociated DRG neurons, which are grown for 21 DIV in NGF (previously treated with FUdR also from 1DIV-5DIV). After 21 DIV, I stop adding NGF for a week before adding embryonic OPCs, which were differentiated from cultured neural stem cells, to the bare axons. Working with 12 mm coverslips, I add 50-100k OPCs and allow them to differentiate (with T3).
Generally the initial seeding of cells looks great, but at about 5DIV coculture, there appears to be a massive die off. In the end I get a culture that appears to be mostly astrocytes with very few MBP positive oligos.
Sometimes "islands" are found with about 10-30 oligos myelinating nicely, but I need the culture to be more uniform.
I have tried seeding them in multiple media types, such as medium supporting NSC growth, OPC growth, and oligodendrocyte differentiation
None of our cultures are done with FBS, but I have tried 1% FBS before, without much luck. Please feel free to ask any further questions and I appreciate any help given.
Interesting. As a first look, OPCs' number looks high. Are those isolated or isolated and cultured? I used CNTF and T3 in the differentiation medium. But i worked only with OLGs.
Neural stem cells are isolated then differentiated into OPCs in a culture. These cells are then lifted and added to neurons. Chan protocol says to use 200k OPCs, but I'm not sure what size coverslip he uses. I have used CNTF also and the OPCs differentiated just fine if I just plate them alone (~30% MBP+ in 14 days)
I checked the article. The number you wrote corresponds to an unknown number of coverslips as they divided into different mediums after the first day of co-culturing. (At least four, according to the setup. I suppose the number well over that). They used isolated OPCs (from 7-8 days brain). Those cells are much more ready to differentiate. I would suggest to lower the number your OPCs well. In lower density, OPCs are more ready to diff, anyway.
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