Abstract
Calcium mobilization assay is a cell-based second messenger assay to measure the calcium flux associated with Gq-protein coupled receptor activation or inhibition. The method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. In some cell lines in which organic-anion transporters are particularly active (e.g. CHO, HeLa), addition of probenecid, an inhibitor of anion transport, is required for retention of this dye in the cells. The dye binds the calcium released from intracellular store and its fluorescence intensity increases. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of the receptor of interest. This protocol can be applied to most mammalian cell lines expressing both endogenous and transiently/stably transfected receptors. The method is sensitive enough to be used for low-expressing systems or high throughput screening of target of interest. Note: The method does not differentiate the Ca2+ mobilization induced by Gqα from the Ca2+ mobilization induced by Gβγ.
Keywords: GPCR, Calcium, Gq protein, FLIPR, AT1R
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. HEK293-AT1R cells stimulated with (well H1) and without (well A1) 1 μM Sar1-Angiotensin II at 18th second. The decrease in the signal with time is most likely resulted from receptor internalization.
Recipes
Acknowledgments
This work was supported by the National Institutes of Health grants HL47570 and HL115964 (Sadashiva Karnik, Ph. D.) and National Research Service Award HL007914 (Hamiyet Unal, Ph. D.).
References
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