FLIPR Calcium & Calcium 3, 4 & 5 Assay Kits

The FLIPR line of Calcium Assay Kits offers the broadest selection of calcium mobilization assays to address diversified biological targets.

Whether you're conducting assay development, running high-throughput screens, or performing basic research there's a FLIPR®  Calcium Assay Kit that meets your assay needs. With four different assay kits, Molecular Devices has the broadest selection of calcium mobilization assays to address diversified biological targets.

FLIPR Calcium Assay Kits combine fluorescent calcium sensitive dyes which measure changes in intracellular calcium, with patented quench technology that minimizes background fluorescence.  Measurement of intracellular calcium occurs via a homogeneous (no-wash) assay method that optimizes cellular response and increases cell-based high-throughput screening. 

The assay requirements for rapid kinetics of calcium signaling events call for validation and optimization of the assay to ensure data quality. FLIPR Calcium Assay Kits are optimized and validated for use with the FLIPR^TETRA® System and take advantage of its proven ability to simultaneously pipette and read to capture the rapid kinetics of calcium signaling, while ensuring you have confidence and reliability in your assay data.

Key Assay Features and Benefits

• Homogeneous (no-wash) assay method enables maximum fluorescent signal from a wide range of cell types, by reducing cell wash artifacts.
• Reduced well-to-well variability improves the quality (Z'-factor) and reliability (CV %) of high-throughput screening assays.
• Universal mix-and-read protocol accelerates assay workflow and increases throughput.
• Superior signal-to-noise ratio facilitates confirmation of transiently transfected receptor activity during assay development.

Four kit formulations are available to address a dynamic range of receptor targets and types of cell lines. Among the many biological targets that have been successfully tested are chemokines; chimeras and promiscuous-coupled receptors; ligand gated calcium channels; and receptors for small peptides, orphan, endogenous, and low-expression receptors. For more information please visit the FLIPR Calcium Assay Description tab.

Workflow

Description

Data

Compatibility

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FLIPR Calcium Kits provide a homogeneous assay for GPCR drug discovery:

FLIPR Calcium Kits include a calcium sensitive dye that is taken into the cytoplasm of the cell during incubation.  The kit's masking technology remains outside the cell and blocks background fluorescence. Upon ligand binding to the receptor, calcium is released into the cytoplasm of the cell.  The dye binds to the intracellular calcium and becomes fluorescent.

 

Choose the Right FLIPR Calcium Assay for Your Specific Biological Target

There are four FLIPR Calcium kit formulations available.  These formulations are specifically designed to address a broad range of targets, including challenging receptors such as chemokines, receptors for small peptides, as well as transient, endogenous, and low-expression receptors which have been difficult to assay with methods that use wash protocols.

Calcium Assay	Product Description
Calcium Kit Link to product brochure	First homogenous no wash kit incorporating calcium sensitive fluorophore and proprietary quench technology designed for robust calcium flux assays such as the response given by agonism of the Muscarinic M1 receptor.
Calcium 3 Kit Link to Product brochure	Improved signal window with new fluorophore enables detection of chemokines especially.   Quench technology designed for targets that have proteins or peptides as agonists.
Calcium 4 Kit Link to product brochure	Contains an updated proprietary masking dye with higher light extinction in the extracellular solution which significantly reduces background and increases signal window.  It is more inert and reduces compound interference. Successful targets include calcium channels, Chemokine receptors, and transient transfections.
Calcium 5 Kit Link to product brochure	A new, novel fluorophore provides maximum signal assay window in conjunction with reduced background capabilities of the superior masking technology used in the Calcium 4 kit.  In addition to the above assays, receptors with low receptor expression and other targets that give a small calcium response are candidates for this kit.

Patented technology

FLIPR Assay Kits from Molecular Devices employ a quenching dye to reduce background fluorescence and improve the signal-to-noise ratio. The patented quench technology (U.S. patent number 6,420,183, EPO patent number 0 906 572) is offered to drug discovery and life science researchers exclusively by Molecular Devices, through the purchase of FLIPR Assay Kits.

Molecular Devices has the broadest selection of calcium mobilization assays to address diversified biological targets. Click on the link below to view data demonstrationg  the benefits of each kit for your assay. In each section, are additonal posters and application notes.

• FLIPR Calcium Kit No wash assay increases well-to-well reproducibility
• FLIPR Calcium 3 Kit Superior results from chemokines and small peptide targets
• FLIPR Calcium 4 Kit Novel masking dye enhances signal to noise ratio
• FLIPR Calcium 5 Kit Masking dye and novel calcium ionophore demonstrates the largest signal window

Calcium 5 Assay Kit Demonstrates Biggest Signal Window Over Other Assay Kits

Figure 1: Comparison of fluorescent signal response to calcium flux during acetylcholine agonism of Muscarinic M1 receptor in CHO M1 cells.  This illustrates the signal-to-noise ratios offered by various FLIPR Calcium Kits with quench technology. Combining a novel fluorophore and the proven quench technology of Calcium 4 Kit, the FLIPR Calcium 5 Kit shows the highest signal to background ratio.

Enhanced Performance, Consistently

Figure 2: Antagonism of calcium flux in response to EC80 acetylcholine in CHO M1 cells evaluated with the Calcium 5 Assay Kit and the FLIPR Tetra System, demonstrating a large signal window and performance consistent with previously published data.

Figure 3: Signal trace on FLIPR Tetra System of ATP agonism of PY receptor in HEK 293 cells compared using different calcium mobilization kits.  Consistent EC50 values among all kits were recorded with no incrase in background. The signal window was substantially larger for the Calcium 5 Assay Kit.

FLIPR Calcium 5 Kit Posters:

• Comparison of FLIPR Calcium 5 Assay Kit performance to other kits
• Functional characterisation of recombinant muscarinic M3 receptors in Assay-ready frozen cells using a novel Calcium Assay kit on the FLIPR Tetra instrument
• TRPV1 assay evaluation with Calcium 5 Assay Kit on the FLIPR Tetra instrument

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FLIPR Calcium 4 Assay Kit Novel Masking Dye Enhances Signal to Noise Ratio

Figure 4: Background reduction in the FLIPR Calcium 4 Assay Kit (orange) leads to improved ΔF/Fo values compared to the FLIPR Calcium 3 Assay Kit (green) in an assay with especially small calcium response. Assay: Endogenous response to carbachol in parental HEK cell in a 384-well plate, 10,000 cells/well, media removal before assay on the FLIPR Tetra Instrument.

Figure 5: FLIPR Calcium 4 and Calcium 3 Assay Kits perform in many cases equally well while at the sametime outperforming competitive kits as shown by a Molecular Devices customer in the data at right. Assay: Undisclosed non-adherent cells timulatesd with a protein activator, no media removal, assay was done on a FLIPR 3 instrument.

FLIPR Calcium 4 Kit Posters:

• Calcum 4 Assay Kit performance on the FLIPR Tetra Instrument

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FLIPR Calcium 3 Assay Kit Demonstrates Superior Results from Chemokine and Small Peptide Targets

Figure 6: Comparison of signal intensity obtained with FLIPR Calcium 3 Assay Kit vs. Fluo-4 and Fluo-3 wash protocols. Response of CHO cells transfected with M1-muscarinic receptors to 100 nM carbachol as measured on a FLIPR 3 system using the FLIPR Calcium 3 Assay Kit.

Figure 7: CHO cells stably transfected with CCR5 and GQi5 were seeded overnight in 100 μL per well of a 96-well poly-d-lysine coated plate. The cells were incubated with 100 μL of the FLIPR Calcium 3 Kit for 1 hour at 37 C. The agonist, RANTES, was added to achieve the final indicated concentration (n=4/group).

FLIPR Calcium 3 Kit Posters:

• Calcium mobilization in CHO M1WT3 cells by carbachol stimulation of the Muscarinic M1 receptor using FLIPR Calcium 3 Assay Kit and the FLIPR Tetra instrument

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FLIPR Calcium Assay Kit Demonstrates Superior Well-to-Well Reproducibility

Figure 8: Comparison of FLIPR Calcium Assay kit with Fluo-3 Dye wash assay.  Figures A and B are Signal traces demonstrating HEK 293and CHO-M1 cells evaluated on the FLIPR 3 System following a 1 hour fluo-3 dye loading incubation. Cells underwent numerous wash steps prior to analysis.  Data shows cells stimulated with 10 μM carbachol. Figures C and D represent data obtained following analysis of HEK293 and CHO-M1 cells on the FLIPR 3 system when using the FLIPR Calcium Assay Kit.  Cells were loaded with kit reagents for one hour, immediately transferred to the FLIPR 3 instrument, and expposed to 10 μM carbachol duing analysis.  No washing of the cells occured. The comparison shows that the results from the FLIPR Calcium Assay Kit demonstrated superior well-to-well reproducibility.

FLIPR Calcium Kit Posters and Application Notes:

• FLIPR Calcium Assay Kit on the FLIPR Tetra instrument
• FLIPR Calcium Assay Kit on the FLIPR 1 and the FLIPR 384 instruments

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MOLECULAR DEVICES FLIPR Calcium Kits are compatible with the FLIPR^TETRA System and the FlexStation 3 Microplate system.  For more information, click on the link to the Molecular Devices instrument and reagent compatibility chart.

FLIPR Calcium Kit Product Configuration Chart

Product Description	Product #	Explorer	Bulk	Express	Evaluation
Calcium Kit	R8041	x
	R8033		x
Calcium 3 Kit	R8091	x
	R8090		x
	R8108			x
Calcium 4 Kit	R8142	x
	R8141		x
	R8143			x
Calcium 5 Kit	R8185	x
	R8186		x
	R8187			x
Calcium 3, 4, and 5 Evaluation Kit	R8172				x

Kit Configuration

Explorer	1 vial = 1 plate x 10 vials + Component B buffer
Bulk	1 vial =10 plates x 10 vials
Express	1 vial = 50 plates x 2 vials
Evaluation Kit = Calcium 3, Calcium 4, and Calcium 5 Kit Dyes	1 vial = 1 plate x 3 vials each + Component B buffer
Component B buffer = HBSS + Ca2+, Mg2+, and 20 mM HEPES