increased DeltaF/F0 value, where F0 is the base-line level of fluorescence and Delta F is the change in fluorescence from the baseline level (peak-baseline) for any given trace. This benefits those assays with especially small calcium responses, as illustrated in Figure 1.

Furthermore, the increased light extinction of the masking dye significantly reduces the addition artifact, also called “the dip.” This artifact is primarily caused by the dilution of the non-fluorescent compound into a plate with media-containing fluorescent components (like DMEM media). Figure 2 illustrates the improvement in the signal with Calcium 4 for HEK 293 cells when used without media removal—the litmus test for the dye artifact. In addition, the inert nature of this novel masking dye further reduces potential interference with ligands or targets.

Optimized AssAys

The FLIPR Calcium, Calcium 3, and now Calcium 4 Assay Kits use homogenous mix-and-read, no-wash protocols. These kits accurately and easily detect intracellular calcium flux from different cell types and receptors. The kits offer

A next-generAtiOn nO-WAsh, hOmOgeneOus, cAlcium mObilizAtiOn AssAy designed fOr the mOst chAllenging And difficult gpcr tArgets

flipr calcium 4 Assay Kit

In drug discovery and research environments, Molecular Devices’ calcium flux assay kits, introduced in 1999, have become the platform of choice for measuring GPCR calcium mobilization in cell-based assays. These cell-based, no-wash kits were developed for Molecular Devices’ FLIPR® and FlexStation® systems for GPCR screening and assay development, respectively.

To address the challenging requirements of an expanding number of GPCRs and ligands, Molecular Devices introduces the FLIPR Calcium 4 Assay Kit. This new kit utilizes a novel masking dye technology which is exclusively licensed to Molecular Devices from Bayer AG (patent nos. US 6,420,183, EP 0906572). The masking technology significantly lowers background fluorescence and improves signal-to-noise without washing the cells.

nOvel mAsKing dye

The FLIPR Calcium 4 Assay Kit has greater light extinction in the extracellular solution due to a novel masking dye, which is also more inert compared to the dye used in the FLIPR Calcium 3 Assay Kit. This feature contributes to a significantly lower background and an

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no wash

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labor saving

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time saving

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better results

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cost saving

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higher signal-to-noise ratio

flipr cAlcium 4 AssAy Kit

background reduction in the flipr calcium 4 Assay Kit (orange) leads to improved deltaf/f0 values compared to the flipr calcium 3 Assay Kit (green). Assay: endogenous response to carbachol in parental heK cells using flipr calcium 3 and calcium 4 kits from a 384-well plate, 10,000 cells/well, media removal before assay on fliprtetrA®.

FLIPR Calcium 4 Assay Explorer KitPart Number: R8142> (10) vials Component A*

> (1) bottle Component B* Each reagent vial sufficient for 1 plate (96, 384, 1536)

R8141 FLIPR Calcium 4 Assay Bulk kit> (10) vials Component A*

* Each reagent vial sufficient for 10 plates (96, 384, 1536)

R8143 FLIPR Calcium 4 Assay Express Kit> (2) vials Component A*

* Each reagent vial sufficient for 50 plates (96, 384, 1536)

sAles Offices

> USA +1-800-635-5577> Australia +61-3-9896-4700> Brazil +55-11-3616-6607 > China (Beijing) +86-10-6410-8629 > China (Shanghai) +86-21-6887-8820> Germany +49-89-9605-880> Japan (Osaka) +81-6-6399-8211> Japan (Toyko) +81-3-5282-5261> South Korea +82-2-3471-9531> Norway +47-32-859-800> United Kingdom +44-118-944-8000

Check our web site for a current listing of our worldwide distributors. www.moleculardevices.com

flexstation, flipr, and fliprtetrA are registered trademarks of molecular devices

corporation. All other trademarks are the property of their respective owners.

specifications subject to change without notice.

different formulations to address a broad range of targets, including challenging systems such as chemokines, receptors for small peptides, and transient, endogenous, and low-expression receptors that have been difficult to assay with methods that use wash protocols.

rApid AssAy develOpment

The pre-optimized, homogenous formulations make the FLIPR Calcium Assay Kits less labor intensive to run. With the mix-and-read procedure, the cells can be incubated with the reagents and are stable for several hours. FLIPR and FlexStation systems are optimal instruments to run the FLIPR Calcium Assay Kits for rapid detection and analysis. These kits can be run at room temperature, which is conducive to automation for high throughput screening. Larger unit volume packaging is available in the Express Kit format to minimize dye preparation.

increAsed thrOughput

The FLIPR Calcium Assay Kits increase throughput by eliminating cumbersome wash steps, which can contribute to data variability. Furthermore, common problems such as cell detachment, lowered cell competency and incomplete dye removal are easily avoided. These no-wash assays further eliminate the potential of dispensing and washing errors or associated equipment failures, and further insures the integrity of screening operations.

selecting the ideAl cAlcium AssAy Kit

An Evaluation Kit is available which contains the FLIPR Calcium, Calcium 3, and Calcium 4 products to provide a convenient way to compare the different FLIPR Calcium Assay formulations side by side. An example is shown in Figure 3.

Ordering infOrmAtiOn

FLIPR Calcium Assay Evaluation KitPart Number: R8172> (3) vials Component A of FLIPR Calcium

Assay Kit*

> (3) vials Component A of FLIPR Calcium 3 Assay Kit*

> (3) vials Component A of FLIPR Calcium 4 Assay Kit*

> 1 bottle Component B

* Each reagent vial sufficient for 1 plate (96, 384, 1536)

significantly increased signal-to-back-ground-intensity ratio (figure 1)

FLIPR Calcium 4 Assay enables homo-geneous calcium flux assays without media removal (figure 2)

FLIPR Calcium Assay Kits: better performance (figure 3)

the flipr calcium 4 Assay Kit reduces the addition artifact caused by intrinsic media fluorescence in parental heK cells without media (dmem) removal. Assay: endogenous response to 333 µm Atp in parental heK cells using flipr calcium 3 and calcium 4 Assay Kits, 384-well plate, 10,000 cells/well, no media removal before assay on fliprtetrA.

molecular devices corporation | 1311 Orleans drive | sunnyvale, cA 94089 usA | email: info@moldev.com©2006 molecular devices corporation. printed in u.s.A. 7/06 1K #0120-1425A

flipr calcium 4 and calcium 3 Assay Kits perform in many cases 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 flipr3 instrument.

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25,000FLIPR Ca 4EC50 = 1.7 µg/mLZ’ at EC50 = 0.84

FLIPR Ca 3EC50 = 1.8 µg/mLZ’ at EC50 = 0.88

competitor’s CaEC50 = 2.1 µg/mLZ’ at EC50 = 0.62