When looking for a needle in a haystack, it helps to shed a little light on the subject — especially light of different hues. A recent investigation found that using two calcium reporter dyes could significantly reduce false-positives in cell-based high-throughput screening, which could produce better screening of compounds and therefore improve drug discovery. If all goes as designed during high-throughput screening using a calcium reporter dye, the dye enters a cell, binds to any free calcium mobilized as a result of an agonist activating a receptor and then responds fluorescently when excited at the right wavelength. When screened against a host of compounds, this information can help determine their effect on cells. However, when a calcium reporter dye that emits at a particular wavelength is used, other compounds present also might fluoresce at that wavelength. This results in a false-positive — it looks as though the reporter dye is binding to calcium when it actually is not. Another problem is that the compound could be a quencher — one that inhibits fluorescence. In that case, the reporter dye is bound to mobilized calcium but because of the suppression of its fluorescence, it seems not to be. Two dyes are better than one. In these charts, the effect of quenching from one calcium reporter dye is plotted against another for 3520 compounds after either 53 s (top) or 26 s (bottom). In each chart, data points in the lower left are double negatives with no quenching from either dye. Data points in the upper right are double positives. Quenching of both could indicate that the compound is an antagonist that blocks receptors. However, points in the upper left and lower right quench one dye and not the other and thus might be considered quenchers if only one dye were used. Courtesy of Kelly J. Cassutt, Hamamatsu Corp.The widely used Fluo-3 dye and its close variant Fluo-4 excite near 480 nm and emit at 540 nm. Thus, when these dyes are used, compounds that fluoresce at 540 nm cannot be tested because they would muddy the results. Consequently, some information must be abandoned, or researchers have to live with some number of false-positives.But there is more than one calcium reporter dye, said scientist Kelly J. Cassutt of Hamamatsu Corp.’s System Div. in Bridgewater, N.J. He pointed to Fura-2, which is routinely used in academic studies but not used often in commercial high-throughput screening. It excites at 340 and 380 nm and emits at 510 nm. The difference between its excitation wavelength and that of the Fluo dyes provides a way to circumvent any autofluorescence of compounds under test because the compound may fluoresce at 480 nm but not at 540 nm.To test this idea, he screened an assay of compounds using Fluo-4 along with Fura-2. He used the Hamamatsu model FDSS6000 which, unlike other systems, uses a xenon lamp as a light source instead of a laser. As a result, the instrument can emit excitation wavelengths anywhere from 300 to 700 nm through the use of the appropriate filters and other optics.In all, Cassutt screened a total of 3520 compounds looking for autofluorescence and quenching. To determine autofluorescence, he measured the response change for Fluo-4 and compared that with the response change for Fura-2. Some compounds responded with a marked and rapid increase in fluorescence and with a much steeper slope versus time than that found in the case of cell death. On average, the fluorescence for such compounds increased fourfold. Most of those, however, did not exhibit a fluorescence increase when Fura-2 was used. The one exception, which at first seemed to be a double false-positive, turned out to be a case of cell death and indicated that the compound was highly cytotoxic.To look for quenching, Cassutt loaded cells with either Fluo-4 or Fura-2 and then incubated them with the compounds at 10-μm concentration. After a 30-min incubation, he added an agonist to bind to specific receptors and to trigger a response within the cells. He then measured the percentage of fluorescence inhibition in the 3520 compounds after 26 and 53 s, and plotted the amount of Fluo-4 inhibition against Fura-2.He found that the compounds tested could be divided into four groups. The vast majority were double negatives and thus had little or no quenching effect for either dye or receptor antagonism, which is the blocking of binding to a receptor. Some were double positives and quenched both or, more likely, were true antagonists. The rest yielded positive quenching results but only for one or the other dye, suggesting dye-specific quenching. These results suggest that using two dyes produces better results than one because compounds identified by both likely are true antagonists. “They’re probably more real than we would have otherwise seen using just one reporter dye or the other,” Cassutt said.He added that these results reveal that some compounds show dye-specific inhibition, suggesting dye-specific quenching. These false-positive hits will be identified as such only when another reporter dye or nonfluorescent-based assay uncovers them as putative quenchers.This investigation is complete, but some work has been done to extend the idea. Instead of loading cells in separate wells with either Fluo-3/4 or Fura-2, cells loaded with one of the two dyes have been put in the same well. Again, the results indicated that the dual-dye technique could weed out false-positives. As for the development of the different assays, Cassutt simply switched Fura-2 for Fluo-3/4 in the assays, and thus there was virtually no extra effort in that regard.Contact: Kelly J. Cassutt, Hamamatsu Corp., Bridgewater, N.J.; e-mail: kcassutt@hamamatsu.com.