View All Posters

P2014
An Assessment of Multiple Fluorescence Calcium Indicator Dyes in Three Diverse Recombinant Systems By Measurement in A Flipr Cell-Based Calcium Flux Assay
Presenter Yeung Wai Lock, Wyeth Research, USA
Additional Authors: J. Zhang, K. Chan, A. Kramer, R. Roncarati D. M. Kowal, and J. Dunlop
Fluorescent dye reagents have been widely used as a screening tool to evaluate functional activities in cell-based assays using the Fluorometric Imaging Plate Reader (FLIPR). Recently, the availability of an array of ‘no-wash’ calcium indicator dyes has enhanced the capacity for investigators to readily optimize functional assays utilizing recombinant cell lines for such a cell-based approach. The ‘no-wash’ homogenous platform has also simplified assay development for high-throughput screening campaigns in the identification of potential therapeutic agents. In this study, we have evaluated FLUO-4-AM and five ‘no-wash’ calcium indicator dyes (i.e. BD Calcium, BD PBX Calcium, Calcium 3, Calcium 4 and FLUO-4 NW) and compared the functional agonist responses in three different recombinant systems; the alpha7 nicotinic acetylcholine receptor (a7 nAChR) expressed in rat pituitary tumor-derived GH4C1 cells, the metabotropic glutamate receptor 5 subtype (mGluR5) expressed in HEK293 cells and the oxytocin receptor (OTR) expressed in CHO cells. In GH4C1/a7 nAChR cells, the maximum relative fluorescence units (RFUs) for nicotine stimulatory responses were comparable among the dyes (9517-13710), with the exception of Calcium 3 (5172) where the signal was reduced by approximately 50%. A similar evaluation with the OTR/CHO cell line revealed a greater than 50% reduction in OT stimulatory responses with the FLUO-4-AM wash protocol (14185) compared with activities achieved by the ‘no-wash’ dye reagents (33326-39573). Mean RFUs produced by L-glutamate with HEK293/mGluR5 cells were considered robust for all six dyes (21654-34392). Further evaluation of concentration-response determinations resulted in comparable EC50 values for nicotine (1.2-2.1 mM) and L-glutamate (0.5-1.4 mM) in the a7 nAChR and mGluR5 recombinant systems, respectively, for all six dyes. However, OT was found to be significantly less potent with FLUO-4-AM (EC50= 77 nM) in comparison with dyes utilizing the ‘no-wash’ platform (EC50 range of 1.5-3.0 nM). These data collectively highlight the importance of evaluating the wide range of calcium indicator dyes in order to select the correct reagent for optimal assay performance in cell-based fluorometric assays.