Fig 1: A multiplex immunoassay based on FP-barcoded reporter cell lines.a A basic panel of FP-barcoded reporter cell lines. K530 cells were transduced with different combinations of 4 FPs to produce 16 uniquely FP-barcoded reporter cell lines. The absence/presence of fluorescence from FPs EBFP2, mTurquoise2 (mTq2), mNeonGreen (mNG), and mCardinal (mCar) are designated as four digits of binary barcodes as shown on the right of histograms for each individual cell line. b Demultiplexing of pooled FP-barcoded reporter cell lines by flow cytometry. c The 16 barcoded reporter cell lines were transduced to express human CD4, CD8a, CD86, and CD154 molecules in a shifted pattern relative to FP expression. These cells were pooled and stained with corresponding mouse monoclonal antibodies (as indicated on the top of each histogram) followed by a PE-conjugated anti-mouse IgG antibody. Signals from individual reporter cell lines were demultiplexed as shown in b and the binding by corresponding antibodies were plotted as half-offset histograms. In all cases, the detected expression patterns were consistent with antigen expression by barcoded cells before multiplexing as shown on the right of histograms for each individual cell line. Isotype Ctrl, mouse IgG1, κ-isotype control antibody. Data from one experiment are shown. Data from another independent repeat experiment are shown in Supplementary Fig. 8.