Hello!
I am trying to differentiated SH SY5Y cells using all-trans Retinoic Acid. I kept the cells for 7 days with 10uM RA (in one experiment I had 3%FBS in medium, and in other 10%FBS). Cells looked different after RA-treatment - I think I could see neurites outgrowth, but when I tried to confirm differentiation using anti-NSE antibodies (NSE - neuron specific enolase - mature neurons marker) I didnt see increasing in NSE level in RA-treated cells in comparing to cells untreated with RA.
What antibodies do you use to confirm differentiation? I have seen some papers, but people often do not agree in case of many markers. Do you have any experience, did you use any differentiation marker FOR SHSY5Y, which shows a big change in level on WB after RA differentiaion?
For neurons, you can use Tuj1(beta 3 tubulin) and map2 markers and to show difference from neural stem cell phenotype nestin and rc2 could be useful. And i agree with the neun stain.
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