Key Assay Features and Benefits

• Maximizes fluorescent signal from a wide range of cell types by reducing cell wash artifacts
• Reduces well-to-well variability, improving assay quality (Z'-factor) and reliability (CV %) of high-throughput screens
• Universal mix-and-read protocol accelerates assay workflow and increases throughput
• Superior signal-to-noise ratio facilitates confirmation of endogenous or transiently transfected receptor activity during assay development
• Pre-optimized and validated protocols for the FLIPR^® Tetra and FlexStation^® Systems ensure you can navigate both routine and unconventional cell lines and targets

FLIPR Calcium Assay Kits Provide a Homogeneous Solution for Assessing GPCRs and Ion Channels

Select the Right FLIPR^® Calcium Assay Kit for Your Biological Target

Choose from six formulations to complement your target of interest, including challenging chemokine, small peptide, transient, endogenous, and low-expression receptors.  FLIPR Calcium Assay Kits are compatible with both direct and indirect targets that trigger intracellular changes in calcium, including GPCRs (Gq or Gα16) and ion channels.

Patented Technology

FLIPR Assay Kits from Molecular Devices. LLC employ a quenching dye to reduce background fluorescence and improve the signal-to-noise ratio. The patented quench technology (more information on patents can be found here http://www.moleculardevices.com/productpatents) is offered to drug discovery and life science researchers exclusively by Molecular Devices, through the purchase of FLIPR Assay Kits.

Molecular Devices offers the broadest selection of calcium mobilization assays to address diversified biological targets. Click on the links below to view data demonstrating the benefits of each assay kit, plus links to additional posters and application notes.

• FLIPR^® Calcium Assay Kit - No wash assay increases well-to-well reproducibility
• FLIPR Calcium 3 Assay Kit - Superior results from chemokines and small peptide targets
• FLIPR Calcium 4 Assay Kit - Unique masking dye enhances signal to noise ratio
• FLIPR Calcium 5 Assay Kit - Novel ionophore demonstrates larger signal window, at a cost effective price
• FLIPR Calcium 6 & 6-QF Assay Kits -  Largest signal window available enabling low signal screens, probenecid- and quench-sensitive assays

FLIPR Calcium 6 Assay Kit Demonstrates Biggest Signal Window and Greater Flexibility Over Other Assay Kits

Figure 1: Comparison of fluorescent signal response to calcium flux during carbachol agonism of Muscarinic M1 receptor in CHO M1 cells. This illustrates the signal-to-noise ratios offered by homogenous assay run in media using the FLIPR Calcium 6 Assay Kit. Combining a novel fluorophore and the proven quench technology, the FLIPR Calcium 6 Assay Kit shows the highest signal to background ratio.

Figure 2: When performing no-wash assays (initial media removal, replaced with assay buffer, no-wash after dye-loading) the novel fluorophore of FLIPR Calcium 6 Assay Kits demonstrates highest signal to background ratio. Obtain same high quality performance in presence or absence of masking dye.

Figure 3: Novel fluorophore is more resistant to organic anion exchange proteins found in some cell lines. No signal seen when using FLIPR Calcium 5 Assay Kit without probenecid (Ca5 Kit no PBX). With FLIPR Calcium 6 Assay Kit (Ca6), the signal is smaller without probenecid, however EC50s are conserved and Z' still greater than 0.5.

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FLIPR Calcium 5 Assay Kit Demonstrates Greater Signal Window than Previous Assay Kits

Figure 4: 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 Assay Kits with quench technology. Combining the novel fluorophore and the proven quench technology of the FLIPR Calcium 4 Assay Kit, the FLIPR Calcium 5 Assay Kit shows higher signal to background ratio than prior kits.

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

Figure 6: Signal trace on the 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 increase in background. The signal window was substantially larger for the FLIPR Calcium 5 Assay Kit.

FLIPR Calcium 5 Kit Posters:

• Comparison of FLIPR Calcium 5 Assay Kit performance to other kits
• Functional characterization of recombinant muscarinic M3 receptors in Assay-ready frozen cells using a novel FLIPR Calcium Assay Kit on the FLIPR Tetra Instrument
• TRPV1 assay evaluation with FLIPR 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 7: Reduced background 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 HEK 293 cells in a 384-well plate, 10,000 cells/well, media removal before assay on the FLIPR Tetra System.

Figure 8: In many cases FLIPR Calcium 4 and Calcium 3 Assay Kits perform equally well while at the same time outperforming competitive kits as shown by a Molecular Devices customer in the data above. Assay: Undisclosed non-adherent cells stimulated 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 9: 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 10: 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 Assay 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 11: Comparison of FLIPR Calcium Assay Kit with Fluo-3 dye wash assay on the FLIPR 3 System. Figures A and B are signal traces demonstrating HEK 293 and CHO-M1 cells 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 when using the FLIPR Calcium Assay Kit. Cells were loaded with kit reagents for one hour, immediately transferred to the FLIPR 3 Instrument, and exposed to 10 μM carbachol during analysis. No washing of the cells occurred. 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 FLIPR 384 Instruments

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Molecular Devices FLIPR^® Calcium Assay Kits are compatible with the FLIPR Tetra and FlexStation^® 3 Systems.  For more information, click on the link to the Molecular Devices instrument and reagent compatibility chart.

FLIPR^® Calcium Kit Product Configuration Chart

Assay Kit Configuration