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

By Brian Schlag and John Dunlop of Neuroscience Discovery Research, Wyeth Research, CN-8000, Princeton, NJ 08543.



SUPERIOR PERFORMANCE

HIGHER THROUGHPUT

IMPROVED DATA QUALITY

RESCUES SUB-OPTIMAL ASSAYS

IMPROVED FUNCTIONALITY IN TRANSIENT TRANSFECTIONS


ABSTRACT
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.


INTRODUCTION
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 typically 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 generation of stable cell lines to confirm functional receptor expression from the plasmid construct, in assay development and validation, and in some cases for compound screening. The results presented highlight an attractive feature of the Calcium 3 no-wash reagent compared with a conventional dye loading and wash protocol for these non-optimal calcium responses. Use of Calcium 3 improved data quality as evidenced by an increase in signal dynamic range, improvement in well-to-well variability and virtual elimination of dilution artifact following compound addition.


METHODS
Cells
HEK-293 cells were used for the transient expression of the 5-HT2C (VSV isoform, tagged at the C-terminus with GFP), 5-HT1A, 5-HT6 or 5-HT7 receptor subtypes. The 5-HT2C receptor couples primarily via Gq to the activation of phospholipase C with subsequent elevation of intracellular calcium. Calcium signaling with the normally Gi-coupled 5-HT1A or Gs-coupled 5HT6 and 5-HT7 receptors was facilitated by transient co-expression with chimeric or promiscuous G-proteins. For transient expression, HEK cells seeded at an approximate density of 3x106 in 10 cm2 dishes were transfected using the Lipofectamine Plus reagent and 24 hours following transfection were re-plated into poly-D-lysine coated 96-well black wall clear bottom plates at a density of 60-80,000 cells per well and used the next day for FLIPR assays.

FLUO-4 FLIPR studies
5-HT stimulated calcium responses were measured using the FLIPR system. Growth medium was removed by aspiration and cells were washed twice with 150 l Hanks buffered saline solution supplemented with 20 mM HEPES and 2.5 mM probenecid (HBSS). Dye loading buffer comprised FLUO-4-AM at 2 M final concentration, 1% fetal bovine serum and 0.1% pluronic acid. After washing, cells were loaded with the calcium indicator dye FLUO-4AM reagent for 60 minutes at 37C. Subsequently, cells were washed once with HBSS at room temperature, 180 l HBSS was added to each well and plates were transferred to FLIPR for acquisition of calcium images. Excitation at 488 nm was achieved with an Argon ion laser and a 515 nm emission filter was used. In each experiment baseline fluorescence after dye loading was adjusted to 10,000 fluorescence counts by adjusting the strength of the laser. Fluorescence images and relative intensities were captured at 1 second intervals and cells were stimulated by addition of agonist (20 l) after 10 baseline measurements.

Calcium 3 FLIPR studies
Growth medium was removed by aspiration, 180 l Calcium 3 reagent, reconstituted in HBSS, was added and cells were loaded for 60 minutes at 37C. Subsequently, plates were transferred to FLIPR for acquisition of calcium images.


RESULTS
Calcium responses to a maximally effective concentration of 5-HT (10 M) in HEK cells transiently expressing the 5-HT2C (VSV isoform) receptor measured using either Calcium 3 or FLUO-4 are presented in Figure 1. In all data examples shown, cells were seeded at modest over-confluence at the time of transfection. That the results obtained for the 5-HT2C transfection are sub-optimal is evidenced by data from other experiments where maximum fluorescence counts in the range of 8,000-12,000 can be achieved with FLUO-4 using HEK cells transfected with the same construct under optimum cell seeding conditions. (See Figure 1, inset.) Figure 1 illustrates the advantage of using the Calcium 3 no-wash reagent under sub-optimal experimental conditions yielding a higher maximum signal in response to agonist, lower standard errors and improved well-to-well variability and elimination of the dilution artifact observed with FLUO-4 following agonist addition. In addition, under optimal cell seeding conditions Calcium 3 yielded greater sensitivity and less variability than FLUO-4, although both reported robust calcium transients. (See Figure 1, inset.)


Figures 2-4 present results from similar comparisons between Calcium 3 and FLUO-4 for the 5-HT1A, 5-HT6 and 5-HT7 receptors transiently expressed in HEK cells. Calcium signaling was facilitated with the 5-HT1A and 5HT6 receptors by co-expression with a chimeric G-protein, and the 5-HT7 receptor was coexpressed with a promiscuous G-protein. Under optimal experimental conditions responses in the range 6000-7000, 5000-6000 and 2000-4000 have been measured with FLUO-4 for 5-HT1A (Figure 2, inset), 5-HT6 and 5-HT7 (not shown) transient expression, respectively. In the 5-HT1A example (Figure 2), use of Calcium 3 resulted in an improved assay performance similar to that described above for the 5-HT2C receptor, i.e., specifically enhanced signal, less variability and reduced dilution artifact. Under optimal conditions, Calcium 3 and FLUO-4 exhibited equivalent performance both in terms of signal range and variability (Figure 2, inset). In the case of the 5-HT6 (Figure 3) and 5-HT7 (Figure 4) receptor evaluations, the difference in assay performance attributable to use of Calcium 3 following non-optimal cell seeding conditions was much more dramatic. Robust calcium transients were measured in the assays run with Calcium 3, while profiles obtained with FLUO-4 were subject to a large dilution artifact and small signal range in response to agonist stimulation.


SUMMARY
A number of fluorescent indicator dyes, including FURA-2, FLUO-3, FLUO-4 and the no-wash reagent FLIPR Calcium and Calcium Plus assay kits, are available for the measurement of changes in intracellular calcium following GPCR activation. FLIPR Calcium 3 Assay Kit (Calcium 3), the newest of the no-wash formulation, outperforms its predecessors and offers sensitivity at least equivalent to FLUO-4, and is broadly applicable across a wide array of GPCR targets.

In the examples shown here where data were acquired with sub-optimal transient transfection conditions, the benefit of the no-wash feature of Calcium 3 is highlighted over the use of a conventional dye loading and wash protocol. Data obtained with Calcium 3 were subject to less variability, higher signal dynamic range and elimination of a significant dilution artifact observed with the conventional dye loading and wash protocol. Calcium 3, a sensitive and reproducible indicator for intracellular calcium, is a significant addition to the reagent assortment available to investigators.


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