is it necessary to starve cells while using a dual luciferase assay to detect expression on TGF-b cell signaling?
I will perform an experiment and I would like to know the pros and cons of starving cells prior to stimulation with recombinant human TGF-b in HEK293 cells.
Thanks in advance
Salah A. Alshehade
Starving cells prior to stimulation is not required for the dual luciferase assay.
Pros:
- Removes any residual serum/growth factors in the media that could cause background signaling and interfere with the TGF-beta pathway specifically being assayed.
- Can synchronize cells into a resting state to provide a more defined baseline for comparing TGF-beta induced signaling.
Cons:
- Could lead to more cell death.
- Starvation is a stress that could trigger other signaling pathways like mTOR, AMPK etc. This could cause crosstalk and make it hard to discern TGF-beta specific effects.
- Basal metabolism and protein synthesis rates will be lowered, which could reduce the magnitude of signal measured in the assay.
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Skylar King
Hi, we found that it is best to starve cells for at least 2 hours before TGF-b treatment. But generally, I starve overnight.
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