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FLIPR Calcium 3 Assay Kit - An Optimal No-Wash Fluorescence Calcium Indicator Reagent for Non-Optimal Calcium Responses

ypically used for measurements of intracellular calcium signaling are FURA-2, FLUO-3 and FLUO-4. These dyes report increases in intracellular calcium by virtue of changes in their fluorescence signal upon calcium binding. FURA-2 exhibits a spectral shift following calcium binding and is used for ratiometric measurement of calcium concentration with dual wavelength UV excitation and single wavelength emission. On the other hand the visible light-excitable FLUO-3 and FLUO-4 lack a significant spectral shift following calcium binding, restricting their use to assessment of qualitative changes in calcium, yet offering superior sensitivity with larger changes in fluorescence intensity following calcium binding. Additionally, two no-wash calcium assay kits from Molecular Devices, the FLIPR Calcium Assay Kit and the Calcium Plus Assay Kit, are available in support of the Fluorometric Imaging Plate Reader (FLIPR) platform. These reagents have provided a homogenous no-wash assay format as an alternative to the more time consuming FLUO-3 and FLUO-4 wash protocols. Despite the significant advantage of a homogenous assay these reagents have not eliminated the use of FLUO-3 and FLUO-4, which are often more sensitive and yield better responses across a more diverse spectrum of biological targets. FLIPR Calcium 3 Assay Kit (Calcium 3), the most recent addition to the range of no-wash dye formulations, overcomes the limitations of its predecessors, demonstrating wide applicability across a diverse array of GPCR targets and sensitivity at least equivalent to FLUO-4.

In the following examples, several GPCRs were transiently transfected into human embryonic kidney cells (HEK-293) at non-optimal conditions; specifically the cells were seeded at modest over-confluence at the time of transfection. Subsequently, these cells were tested with the Calcium 3 no-wash reagent to measure agonist-stimulated calcium signaling. Transient transfections are frequently used prior to ge
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