Evaluation of FLIPR Calcium 3 Assay Kit--a new no-wash fluorescence calcium indicator reagent.
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 EC(50) values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compelling 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.
Zhang
Yingxin
Y
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA.
Kowal
Dianne
D
Kramer
Angela
A
Dunlop
John
J
D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000032:analysis*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D018698:Glutamic Acid / Q000494:pharmacology; D011933:Reagent Kits, Diagnostic; D064529:Receptor, Metabotropic Glutamate 5; D044405:Receptor, Serotonin, 5-HT2C / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000819:agonists / Q000378:metabolism; D012680:Sensitivity and Specificity; D012701:Serotonin / Q000494:pharmacology; D058826:Serotonin 5-HT2 Receptor Agonists; D014966:Xanthenes / Q000737:chemistry / Q000378:metabolism
D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D011933:Reagent Kits, Diagnostic; D064529:Receptor, Metabotropic Glutamate 5; D044405:Receptor, Serotonin, 5-HT2C; D043562:Receptors, G-Protein-Coupled; D018094:Receptors, Metabotropic Glutamate; D017483:Receptors, Vasopressin; D058826:Serotonin 5-HT2 Receptor Agonists; D014966:Xanthenes; D012701:Serotonin; D018698:Glutamic Acid; D002118:Calcium