FLIPR Calcium Assay Kit

Reagent System for Calcium Flux Assays on FLIPR

• Stronger signals
• Higher assay precision
• Improved physiological data
• No need to wash cells
• Higher sample throughput in fewer hands-on steps

Kit description

FLIPR has become the system of choice for evaluating changes in intracellular calcium in a high throughput-screening environment. Conventional FLIPR assays can be multi-step procedures consisting of a dye-loading step followed by extensive washing of the cells. Several problems are routinely encountered when washing dye-loaded cells, all of which add to experimental variability.

Problems in a conventional assay protocol include:

• Cells removed from the plates during the washing procedure
• Reduced responsiveness (competence) of the cells following washing
• Variation in the residual volume of wash buffer, leading to variation in the concentration of test compound
• The drop in fluorescence (resulting from the dilution of residual extracellular dye) upon addition of the test compound

		Figure 1. Data demonstrates HEK293 cells evaluated on FLIPR following a 1 hour fluo-3 dye loading incubation. Cells underwent numerous wash steps prior to analysis on FLIPR. Data shows cells stimulated with 10 µM carbachol.
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		Figure 2. FLIPR Bitmap displaying data from CHO-M1 cells stimulated with 10µM carbachol. Cells were loaded with fluo-3 for 1 hour, followed by extensive washing prior to FLIPR analysis.
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Optimized assay

The Calcium Flux Assay Kit from Molecular Devices Corporation was developed and optimized for FLIPR to provide a fast, simple and reliable fluorescence based assay for detecting changes in intracellular calcium. With this kit, calcium assays on FLIPR become a Mix and Read procedure in which cells are incubated with the kit reagents for up to one hour and transferred directly to FLIPR for evaluation. There are no intermediate wash steps involved.

Advantages of the Calcium Flux Assay Kit include:

• Higher maximum signal with lower variance across the plate
• Improved physiological data
• Rapid procedure with less hands-on time
• Fewer steps resulting in higher sample throughput
• Ease of use with both adherent and non-adherent cell types
• Cost savings

Superior data

The major benefit of the optimized assay is apparent in the quality of the data. The signal obtained with the Calcium Flux Assay Kit tends to be higher than that observed in cells loaded with fluo-3, and at least comparable to that seen with fluo-4 dye-loaded cells (Figure 3). Furthermore, tighter CVs across 96- and 384-well plates are observed (Figure 4).

		Figure 3. Representation of data obtained following FLIPR analysis of HEK293 cells evaluated on FLIPR when using the FLIPR Calcium Assay Kit. Cells were loaded with kit reagents and immediately transferred to FLIPR and exposed to 10 µM carbachol. No washing of the cells occurred.
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		Figure 4. Results demonstrate response of CHO-M1cells to 10 µM carbachol as measured on FLIPR using the FLIPR Calcium Assay Kit. No wash steps were involved in the protocol.
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Higher throughput

An advantage in any high throughput screening campaign is the reduced number of steps in the assay, leading to even greater throughput with FLIPR. The kit procedure simply involves the direct addition of the kit reagents to the cell plate, incubation for 20 minutes to 1 hour and then immediate transfer of the plates to FLIPR for data collection.

Convenience

The Calcium Flux Assay Kit has been demonstrated to work equally well with both adherent and non-adherent cells. In many cases poly-D-lysine coated plates are not required, further lowering the overall assay cost. With the Calcium Flux Assay Kit, non-adherent cells are less likely to lift off the plate and display greater responsiveness due in part to the elimination of multiple wash/centrifugation steps. Users consistently see equivalent and in many cases superior responses to those obtained using the conventional protocol.

Ordering Information

Product Number	R8033	R8041
Product Name	FLIPR Calcium Assay Kit, Bulk	FLIPR Calcium Assay Kit, Explorer
Unit Size	1 Kit provides reagent for 100 plates (packaged as 10 vials)	1 Kit provides reagent for 10 plates (packaged as 10 vials)
Pricing	Volume discount given for quantities of 5, 10, and 50 units	Volume discount given for quantities of 5, 10, and 50 units