By Brian Schlag and John Dunlop of Neuroscience Discovery Research, Wyeth Research, CN-8000, Princeton, NJ 08543.
IMPROVED DATA QUALITY
RESCUES SUB-OPTIMAL ASSAYS
IMPROVED FUNCTIONALITY IN TRANSIENT TRANSFECTIONS
FLIPR Calcium 3 Assay Kit (Calcium 3), the latest in a series of no-wash fluorescence calcium indicator dye reagents from Molecular Devices, exhibits superior performance to the FLIPR Calcium and Calcium Plus Assay Kit reagents. In particular Calcium 3 has broad applicability across a diverse array of biological targets and possesses sensitivity at least comparable to that of FLUO-4. Elimination of washing steps has the major advantage of reduced plate handling and faster assay throughput. In addition, under sub-optimal assay conditions the use of a homogenous no-wash assay has the potential for improvements in data quality and reduced well-to-well variability. Transient expression of G-protein coupled receptors followed by measurement of functional response typically is subject to more variability than the use of stable receptor expressing systems. Accurate seeding density for cells prior to transfection is a major factor influencing the subsequent analysis of the cells. In cases where cells have been seeded at modest over-confluence prior to transfection, the use of Calcium 3 has permitted successful functional screening of the cells with superior results to the use of a conventional dye loading and wash protocol.
Fluorescent dye indicators for intracellular calcium measurements are widely used tools in biology, in particular in the field of G-protein coupled receptor (GPCR) pharmacology and functional characterization. Examples of dyes t