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

Neuroscience Discovery Research, Wyeth Research, Princeton, NJ

Dianne Kowal

Neuroscience Discovery Research, Wyeth Research, Princeton, NJ

Angela Kramer

Neuroscience Discovery Research, Wyeth Research, Princeton, NJ

John Dunlop

Neuroscience Discovery Research, Wyeth Research, Princeton, NJ, Dunlopj{at}wyeth.com

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-HT_2C), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT_2C) 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-HT_2C receptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC₅₀ values 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)

Key Words: FLIPR calcium 3 assay kit • Calcium 3 • FLUO-4 • fluorometric imaging plate reader • 5-HT2C receptor • mGluR5 receptor • vasopressin V2 receptor • calcium signaling • G-protein-coupled receptors

Journal of Biomolecular Screening, Vol. 8, No. 5, 571-577 (2003)
DOI: 10.1177/1087057103257240


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