Evaluation of FLIPR Calcium 3 Assay Kit—A New No-Wash Fluorescence Calcium Indicator Reagent

Abstract

We have evaluated the FLIPR Calcium 3 Assay Kit (Calcium 3), a new no-wash fluorescence calcium indicator dye reagent, for the measurement of agonist-stimulated calcium signaling in cells expressing the serotonin 2C (5-HT2C), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT2C) or superior (mGluR5 and V2) sensitivity to FLUO-4 as indexed by the change in fluorescence counts following agonist application. Assay variability, indexed by CV, using Calcium 3 or FLUO-4 was equivalent with 5-HT2Creceptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC50values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compel-ling alternative to FLUO-4 in the choice of fluorescent dye reagent for studying G-protein-coupled receptors, providing the advantage of a homogenous, no-wash assay format. ( Journal of Biomolecular Screening 2003:571-577)