Integrated, real-time, whole-cell activity profiles in response to FR, EGF (Sigma), CCh (Sigma), and forskolin (Tocris) were monitored with an optical biosensor on the basis of the detection of DMR (Corning Epic System). For DMR detection, CRISPR-Cas9 Gαq/11 KO cells transfected with empty vector control or plasmid encoding Gαqwt or GαqQ209L using the FuGENE transfection reagent were seeded into fibronectin-coated, 384-well biosensor microplates at a density of 15,000 cells per well 24 hours before measurements were made. On the next day (48 hours after transfection), cells were washed with Hanks’ balanced salt solution containing 20 mM Hepes followed by 1 hour of equilibration before the addition of FR and the appropriate drugs (CCh, 30 μM; forskolin, 30 μM; EGF, 50 nM). Cell responses were measured immediately after compound application. All steps, except washing, were performed at 37°C. To quantify the effects of FR, the area under the curve (AUC) of downward-deflected DMR traces within 0 and 3600 s was used and normalized to the maximum negative response, which was set at −100%.

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