I have been using 20% FBS in DMEM with L-Glu and NEAA.
I started off with half feeds, and then eventually transferred the surviving cells to plastic to select for astrocytes. However, they did not stain positive for GFAP. I will use s100b antibody on them next, but I was wondering how to improve cell survival when differentiating to astrocytes. They look more like fibroblasts right now...
Also: I saw that a thermofisher protocol said to use DMEM with N2, glutamax and 1% FBS.
What media should I use?
Should I do half-feeds or just switch to 100% astrocyte diff media right away?
Hello Jiyeon.
Hard to tell anything without knowing your cell culture dishes and exactly what you did.
However, one thing is clear. You should induce neural differentiation for 3~4 day in stem cell culture condition, and then switch to neural basal medium containing N2, EGF, bFGF, and other NFGs in serum-depleted medium to allow differentiation to glia cell lineages and their proliferation, too. It would take nearly a month.
Follow any proper protocols of neural differentiation of ES or iPS cells.
See the links.
https://pdfs.semanticscholar.org/680b/7556d2724aff37ff9b6608176d7917cb6e8f.pdf
https://www.cell.com/stem-cell-reports/pdf/S2213-6711(17)30281-3.pdf
Best wishes.
Thank you! Your advice and these articles are very helpful. Currently I have some survivors using the thermo fisher recipe, but I think I will increase the amount of FBS. I will see the results of my s100b staining on Monday. I appreciate the help!
You may find useful information in the article available through this link:
https://www.nature.com/articles/srep45091.pdf
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