Hi,
Does anybody has an idea how to perform pharmacological long-term Notch stimulation in cell cultures? I want to stimulate Notch signaling in in vitro differentiated astrocytes for 1-2 weeks. I tried Jagged-1 attached to the poly-L-lysine coated TC plate, which many people use, but the effect on Hes5 epression after 2 weeks was quite low, so I think the Jagged-1 might not be stable long enough in the culture medium...
Thanks for suggestions.
Michael
Hi Maureen,
thanks for your suggestions. I would prefer a pharmacological approach. I thought about NICD overexpression, to see whether Notch signaling is at all critical for the process I am studying, but in principle I want to activate Notch signaling in a kind of in vitro differentiation approach by using some media supplementation. With regard to a possible feedback loop and Hes5 as readout, at least in vivo Notch signaling is active in astrocytes, and the Hes5 levels are much higher than in culture, so it seems it should be possible to get constitutive Notch activation in astrocytes. Also some other described Notch targets show rather low or absent expression in my model. Using directly the soluble Jagged ligand seems to work not too well, as I understand the literature, it seems to need some kind of "membrane"-bound clustered structure to efficiently activate Notch signaling.
Anyway, thanks, I thought I might have overlooked some more straightforward approach, but maybe there is none.
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