March 1, 2006 (Vol. 26, No. 5)

Considerations for Assessing the Performance of Calcium Indicators

G-protein-coupled receptors (GPCRs) are among the most successful targets for drug discovery research. They represent the largest family of cell surface receptors with approximately 400 in the druggable genome. GPCRs are essential in the transmission of a diverse assortment of chemical messages from the extra-cellular environment to the interior of the cell and exhibit potent signaling due to the various second messenger cascades that are activated in response to a stimulus.

Although all GPCRs share certain structural and mechanistic features, the receptors often exhibit different functional responses, even within the same GPCR family, when assayed with different detection chemistries.

Calcium Indicators for Analysis of GPCR Modulation

Intracellular Ca2+ measurements are a cornerstone in high-throughput screening for compounds associated with GPCR modulation. They can distinguish the functional aspects of ligand-mediated receptor activation (via receptor agonists, antagonists, inverse agonists, and allosteric modulators), providing more information than traditional binding-only assays can.

Molecular Probes ( developed the first commercially available calcium fluorophores for analyzing GPCR modulation, including the gold standard calcium indicator fluo-4.

Now, with an increasing number of choices available, an understanding of the practical advantages and disadvantages of each indicator can be a challenge. Different GPCR receptor types and cell types may perform differently and inherently be better suited to different assay development strategies or reagent compositions. Thus, no single, optimal calcium indicator formulation currently exists for all GPCR targets and cell types.

The sensitivity of todays instrument platforms, such as the Hamamatsu FDSS 6000 and PerkinElmers CellLux, has allowed the industry to focus on developing assays with improved performance capabilities, but researchers developing assays for high-throughput screening need to continually consider whether the benefits gained via one product formulation align with their internal goals and capabilities.

Throughput, Convenience vs. Signal

Todays HTS facilities typically utilize sophisticated robotics designed to rapidly and automatically process and analyze tens of thousands of samples per day. In these environments, scientists are under pressure to maximize throughput, even at the cost of achieving the greatest signal intensity.

Minimizing sample intervention/handling (e.g., wash steps and/or media removal) is one means to increase throughput. Therefore, laboratories are increasingly implementing commercially available no-wash calcium indicator formulations such as Invitrogens ( Fluo-4 NW Calcium Assay.

To further increase throughput scientists may elect to eliminate the media removal step in no-wash formulations, compromising signal to increase efficiency. Alternatively, when maximizing throughput is not a key consideration, a researcher can use the Fluo4-NW Calcium Assay and incorporate a wash step or use fluo-3 or fluo-4 to generate higher quality data for a broad variety of GPCR targets.

Achieving Desirable Sensitivities

Early calcium indicator assays often faced challenges in generating enough signal to develop a robust assay and justify investing in a large high-throughput screen of the desired target. Therefore, assay development techniques often required cells to be washed in order to remove extracellular dye, which often contributed to unacceptably high background fluorescence. However, washing typically disrupts attached cells and can yield lower signals, higher well-to-well variability (%CVs), and sometimes presents additional hindrances to throughput.

Newer developments to overcome these obstacles and reduce baseline fluorescence generated new formulations (e.g., Calcium-3), which included the use of background suppression agents. However, such suppression agents can interfere with receptor biology in calcium assays. As a result, researchers have asked for formulations with enhanced performance characteristics that can be used without suppression agents.

The Fluo4-NW Calcium Assay, which results in equal or larger calcium signals in a variety of cell types (both adherent and suspension), enables researchers to analyze and screen Ca2+ cell-signaling events without potential, nonspecific interactions that can be caused by addition of a quenching dye. This new formulation achieves the larger fluorescence intensities that are desired with the convenience of a no-wash protocol that helps lower the %CVs and improve the quality of data (Figure 1).

Fig. 1: CHO M1 Dose Response Curve. Cells were stimulated with carbahol over the indicated concentration ranges. The data shows that the Fluo-4NW assay yielded a greater magnitude of response while displaying similar EC/50 values to other assay methods

Protocol Differences

The loading of calcium indicators, such as Fluo-4NW or Fluo-4AM dyes, into cells is a dynamic process involving cellular uptake of the dye, cleavage of the acetoxymethyl protecting groups by intracellular esterases, and binding of intracellular calcium to the fluorophore. This uptake of dye is counterbalanced with extrusion of intracellular active dye, which contributes to background fluorescence. Time, temperature, and assay buffer formulations all influence the rate of dye uptake and extrusion, affecting signal intensity as well as background.

Therefore, comparing indicators without considering optimization conditions may compromise the performance of some calcium indicators. For example, many calcium indicator protocols consist of a dye-loading recommendation of approximately 60 min at 37C. However, in the development of Fluo-4 NW Calcium Assay, we determined that often the maximum signal intensity and minimal background can be better achieved with a two-step incubation consisting of 30 min at 37C followed by 30 min at room temperature.

For many receptors, this change can yield improved performance characteristics. Therefore, optimization of dye-loading parameters should be a part of any assay-development program.

Fig. 2A: Analyzing Assay Performance. Averages of thee responses of CHO M1 cells to 200 nM carbachol are displayed as raw data in relative fluorescence units

Analyzing Assay Performance

Data analysis of calcium-flux measurements generally are determined as either the net change in fluorescence signal (typically referred to as DF or max signal-background) or the net change in fluorescence signal over background (DF/Fo or S:B).

The DF/Fo calculation has historically been used for comparing data from different labs (assay development done at one site and HTS done at another site), or with assays performed using different instruments.

Although this method is valid for comparisons between different equipment or lab environments, if a researcher is actually comparing the performance of various calcium indicator reagents on a GPCR of interest using a specific instrument, a more direct measure to use would be DF, which is easily calculated and understood.

Because researchers are most interested in the modulation of GPCR activity, performance analysis should be focused on the response (DF), which is the most direct indication of the second messenger cascade. Furthermore, this measurement doesnt obscure the data analysis by dividing by the baseline fluorescence, which is largely due to extracellular fluorescence and has no relation to the GPCR activity.

If one does use DF/Fo when interpreting results, it may be unclear whether a smaller value is due to smaller response (DF) or higher baseline (Fo). Indicator formulations that do not include a background suppression agent, such as Fluo-4NW Calcium Assay, may yield higher background fluorescence, potentially resulting in lower DF/Fo than assay formulations containing a background suppressor (for example, Calcium-3).

If scientists use the DF/Fo approach in this type of scenario, they may unnecessarily eliminate a high-performance formulation option because the DF/Fo is lower, even though the DF shows the indicator formulation would yield a robust assay (Figure 2).

HTS of GPCRs using calcium indicators is an essential method for drug discovery for many years to come. Newer calcium indicator formulations, such as Fluo-4NW Calcium Assay, present an additional tool that offers improved screening efficiency and reduced risk of nonspecific assay interference. As we continue to develop even more new calcium-indicator formulations for specific receptors, cell types, and experimental requirements, a deeper understanding of the best use of these valuable tools will be critical for navigating the challenges faced in drug discovery.

Fig. 2B: Averages of three responses of CHO M1 cells to 200 nM carbachol are displayed as divided by the baseline fluorescence

Fig. 2C: Baseline subtracted.

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