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FLIPR® Calcium Assay Kits are the platform of choice for measuring changes of intracellular calcium for drug discovery and basic research. Built as the most comprehensive portfolio of calcium reagents, the kits deliver pre-optimized, homogeneous, fluorescence-based formulations to expedite assay development and screening of GPCR and ion channel targets.


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FLIPR® Calcium Assay Kits are the platform of choice for measuring changes of intracellular calcium for drug discovery and basic research. Built as the most comprehensive portfolio of calcium reagents, the kits deliver pre-optimized, homogeneous, fluorescence-based formulations to expedite assay development and screening of GPCR and ion channel targets.

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 6 and 6-QF Assay Kits

This exclusive fluorophore features the highest quantum yield of any calcium indicator on the market, delivering the greatest signal window available in a calcium assay kit. The substantial increase in signal amplitude helps to rekindle low signal screens; including endogenous, primary or stem cell targets. Additionally, the novel dye is unaffected by organic anion exchange proteins, enabling measurement with minimal to no probenecid present.

When utilized in the quench-free (QF) formulation, the dye is ideal for quench sensitive targets or assays requiring multiplexing of calcium-flux and luminescent assays within one well.

FLIPR Calcium 5 Assay Kit

A novel fluorophore that amplifies the assay signal window while leveraging the superior masking technology used in the FLIPR Calcium 4 Assay Kit to reduce background fluorescence. 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.

FLIPR Calcium 4 Assay Kit

Contains a revised proprietary masking dye with higher light extinction in the extracellular solution to significantly reduce background and increase signal window. It is more inert and reduces compound interference. Successful targets include calcium channels, chemokine receptors, and transiently transfected receptors.

FLIPR Calcium 3 Assay Kit

Improves signal window with unique fluorophore that enables detection of chemokines. Quench technology designed for targets that have proteins or peptides as agonists.


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 is offered to drug discovery and life science researchers exclusively by Molecular Devices, through the purchase of FLIPR Assay Kits.

Patent Information

Molecular Devices offers the broadest selection of calcium mobilization assays to address diversified biological targets.

  • FLIPR® Calcium 6 & 6-QF Assay Kits – Largest signal window available enabling low signal screens, probenecid- and quench-sensitive assays
  • FLIPR Calcium 5 Assay Kit – Novel ionophore demonstrates larger signal window, at a cost effective price
  • FLIPR Calcium 4 Assay Kit – Unique masking dye enhances signal to noise ratio

FLIPR Calcium 6 Assay Kit demonstrates biggest signal window and greater flexibility over other assay kits


Figure1

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.


Figure1

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.


Figure1

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.


FLIPR Calcium 5 Assay Kit demonstrates greater signal window than previous assay kits


Figure1

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.


Figure1

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.


Figure1

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 4 Assay Kit novel masking dye enhances signal-to-noise ratio


Figure1

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.


Figure1

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 Assay Kits provide a homogeneous solution
for assessing GPCRs and Ion Channels



Figure 1: FLIPR Calcium Assay Kits utilize a calcium sensitive dye that is absorbed into the cell’s cytoplasm during incubation. Upon ligand-receptor binding, intracellular calcium is released, binding with the dye, thereby increasing fluorescence intensity. All FLIPR Calcium Assay Kits utilize an extracellular masking technology to block background fluorescence and increase your assay’s signal window. The FLIPR Calcium 6-QF Assay Kit is the one exception, enabling assay performance without a masking technology, making it an ideal choice for quench-sensitive applications such as multiplexing with luminescent indicators.

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