Double-Color Fluorescence In Situ Hybridization with RNA Probes

914 BioTechniques Vol. 34, No. 5 (2003) amount of tryptase that is spontaneously released from cells. Figure 3 shows dose responses of tryptase release by anti-IgE at various IgE concentrations. Maximal activation of the cells occurred with 0.1 μg/mL IgE with no detectable activation at 0.0001 μg/mL IgE (Figure 3). The EC50 values for anti-IgE were 51 ± 15, 56 ± 7.3, 145 ± 60, and 167 ± 43 ng/mL, respectively, for 1, 0.1, 0.01, and 0.001 μg/mL IgE (Figure 3). When the data were analyzed in a reciprocal manner, the EC50 values for IgE were 1.7 ± 0.3, 2.3 ± 0.4, 3.4 ± 0.3, 5.1 ± 0.8, 7.9 ± 1.8, 17.6 ± 3.2, and 26 ± 7.8 ng/mL, respectively, for 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 μg/mL anti-IgE. A real-time tryptase release assay has been developed. The profile of inhibition of the proteolytic activity released by these cells is consistent with tryptase activity. Tryptase is released by activation of FcεRI in a timeand dosedependent manner (Figures 2 and 3). This homogeneous real-time assay requires small numbers of cells, few processing steps, and offers the possibility of high-throughput screening because it is formatted in 96 wells. In addition, this assay allows for the simultaneous identification of both inhibitors and activators of mast cells without alteration of the assay format. The initial lag in activity (Figure 2) allows for the identification of activators when compounds are pre-incubated with cells. Using this real-time assay, the EC50 values for both IgE and anti-IgE for release of tryptase from CBMCs were determined. Additionally, the maximal amount of tryptase released through FcεRI was compared with that released by calcium ionophore (data not shown). These amounts were equivalent, which is consistent with previous studies (12) in which the percent histamine release through FcεRI was found to be 100% of that released by ionophore from cells cultured in IL-4. The amount of tryptase released in our studies was typically 10%–20% of total tryptase consistent with 10%–20% histamine release from the same cultures. In addition, this assay has been used to examine tryptase release mediated by endothelin-1, platelet activating factor, phorbol ester, and ATP (data not shown). This methodology should also be applicable to mast cells isolated from other species; however, the protease profile of these mast cells will have to be characterized. This assay will be useful to screen for modulators of tryptase release from CBMCs and will allow the identification of both inducers and inhibitors of release.