Multiplexing of pathway-specific beta-lactamase reporter gene assays by optical coding with Qtracker nanocrystals

Reporter assays are widely used in research and drug discovery for analysis of signaling pathways in a cell-based format. Traditionally, reporter gene assays are run in a single-parameter mode, interrogating only 1 pathway per sample. To enable more complex assay formats for pathway analysis, the authors developed a multiplexed reporter cell-based assay that combines optical encoding with a beta-lactamase reporter gene readout. The optical encoding is achieved by peptide-mediated delivery of quantum dots into reporter cell lines. Using different quantum dots, the authors were able to simultaneously analyze multiple signaling pathways in the same sample using fluorescence microscopy or flow cytometry. They selected 3 beta-lactamase reporter cell lines for the analysis of tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and interferon gamma (IFN-gamma) induced signaling to perform proof-of-principle experiments. The analysis demonstrates that this multiplexed assay allows the reliable detection of ligand-specific activation patterns as well as pathway-specific inhibitors. This method provides a template for the development of novel assay designs that enable the analysis of complex signaling networks involving multiple signaling pathways as well as cell-specific pathways in heterotypic cell models.