Dear All,
I differentiate some new clones of hiPSCs into neuronal progenitors using dual SMAD inhibitor in DMEMf12/neurobasal mix+ NEAA +n2+b27.
After induction of 12 days in culture and expanding them in fgf2 contaning medium 2-3 days ; I am able to induce them into mature neurons (they became neun+)
But when I stain my NPCs with Sox2, pax6, foxg1. They are all negative. I get a staining with nestin only. I am not sure with the stage of my cells.
Does anyone have any idea? normally I would expact sox2 pax6 negative cells not to be passaged and differentiated into neurons. But I am able to proliferate them as well.
thanks
yagiz
probably some antibody problems for staining (I assume you performed ICC). as it is nearly impossible for those txn factors to be absent, I'd recommend to troubleshoot staining problem (if any) first and then to try western blottinng or just a simple RT-PCR.
one another thing I'd recommend is to perform stainings no later than day 7-8 where pax6 would potentially be maximized via dual smad inhibition.
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