Nine days after sorting, healthily proliferating cell clones were transferred into 24-well plates for further expansion

Nine days after sorting, healthily proliferating cell clones were transferred into 24-well plates for further expansion. for characterizing immunity and discovering antibodies to membrane-associated proteins. by CRISPR-Cas9-mediated gene targeting (Supplementary Fig.?1). Using genomic DNA sequencing to ensure disabling all alleles, we generated a cloned, basal cell line, K530. Compared with the parental K562 cells, K530 cells are negative for all Fc receptors CD16, CD32, and CD64, and show minimal nonspecific binding by human IgG1 (Supplementary Fig.?1). Stable barcoding of reporter cell lines with unique combinations of FPs To introduce stable barcodes into the reporter cells for multiplex detection, we conceived a strategy to use combinations of FPs expressed in the cytosol of reporter cells. The growing toolbox of FPs9 coupled with widely used multicolor flow cytometry allows for marking and detecting multiple FPs simultaneously. The number of unique FP combinations is 2where is the number of different FPs. This exponential result provides the power for multiplexity. After careful selection and experimental tests, we selected eight FPs, including EBFP210, mTurquoise211, mNeonGreen12, mCardinal13, mKate214, miRFP70315, LSSmOrange16, and hmKeima8.517 (Supplementary Table?1). These FPs are characterized by the following: (1) bright fluorescence for good separation and limited spill-over into other fluorescence channels; (2) good photostability and low cytotoxicity; and (3) monomeric FPs to avoid potential F?rster resonance energy transfer events between A-366 heterologous FPs. We designed and generated a four-color basic panel (Supplementary Figs.?2C5) capable of 16 distinct FP combinations. Extended panels with two (Supplementary Figs.?2 and 3) or four additional colors (Supplementary Figs.?4 and 5) could expand the multiplexity to 64- or 256-plex, respectively. Alternatively, increased multiplexity could be achieved by introduction of A-366 a reference membrane protein (e.g., CD8a) or by high/low intensity versions of the same FPs (Supplementary Figs.?6 and 7). We validated the four-color basic panel in supporting A-366 16-plex detection of cell surface molecules. K530 cells were engineered to express all 16 combinations of four FPs from the basic panel, resulting in 16 uniquely FP-barcoded reporter cell lines (Fig.?1a and Supplementary Fig.?8). Pooled cell lines can be demultiplexed by flow cytometry based on patterns of FP expression (Fig.?1b and Supplementary Fig.?8). The growth rates of these 16 FP-barcoded reporter cell lines were determined individually and after pooling; similar growth rates were observed A-366 in both conditions (Supplementary Fig.?9). Although proliferation rates of all barcoded cells are similar, variation in growth rates is sufficient such that expansion of pooled cell lines should be limited to three to four doublings or Rabbit Polyclonal to Smad1 (phospho-Ser465) about 3 days of culture to preserve comparable and adequate numbers for the reliable detection of each sub-population. To validate the deconvolution of multiplexed cell populations, we generated 16 cell lines expressing human CD4, CD8a, CD86, or CD154 so that each protein was associated with four unique FP patterns. The pooled cells were stained in single tubes with monoclonal antibody for one of the four human antigens, followed by a common phycoerythrin (PE)-conjugated secondary antibody. Deconvolution by flow cytometry showed high resolution of bound and unbound cells and patterns of binding consistent with antigen expression by barcoded cells before multiplexing (Fig.?1c and Supplementary Figs.?8 and 10). Open in a separate window Fig. 1 A multiplex immunoassay based A-366 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.