Simultaneous detection of GPCR second messengers in living cells

Live cell assays constructed with genetically encoded, fluorescent biosensors can provide significant advantages over endpoint assays measured in cell lysates because functional information about the timing and location of cellular responses can be monitored in cells that are relevant to disease. Fluorescent biosensors have been tools in basic research and have now been optimized for detection on automated plate readers. We previously demonstrated that Montana Molecular’s live cell cADDIS assay for cAMP on the CLARIOstar^® produces high Z’ values characteristic of a robust screening assay. Here we show how second messengers relevant to the Gq signaling pathway can be detected in living cells using assays for DAG, PIP2, and Ca²⁺. By combining spectrally distinct sensors in a single assay, we show that two responses can be detected simultaneously. Sensitive detection of these responses is enabled by employing the CLARIOstar^® microplate reader using filters or the LVF monochromator.