Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling


Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5–15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell–receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-γ signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4− T cells and CD11b-hi cells.

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Figure 1: The FCB technique.
Figure 2: Deconvolution methods for fluorescently barcoded samples.
Figure 3: FCB of 36 samples increases throughput of phospho flow.
Figure 4: Small-molecule drug screening in 96-well plate format reveals selective inhibitors.
Figure 5: Signaling profiling in complex heterogeneous populations reveals thermodynamic and kinetic differences in responses to IFN-γ stimulation.


  1. 1

    Tung, J.W., Parks, D.R., Moore, W.A., Herzenberg, L.A. & Herzenberg, L.A. Identification of B-cell subsets: an exposition of 11-color (Hi-D) FACS methods. Methods Mol. Biol. 271, 37–58 (2004).

    Google Scholar 

  2. 2

    De Rosa, S.C., Herzenberg, L.A., Herzenberg, L.A. & Roederer, M. 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat. Med. 7, 245–248 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Krutzik, P.O., Irish, J.M., Nolan, G.P. & Perez, O.D. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin. Immunol. 110, 206–221 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Krutzik, P.O. & Nolan, G.P. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55, 61–70 (2003).

    Article  Google Scholar 

  5. 5

    Fleisher, T.A. et al. Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin. Immunol. 90, 425–430 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D.A. & Nolan, G.P. Causal protein-signaling networks derived from multiparameter single-cell data. Science 308, 523–529 (2005).

    CAS  Article  Google Scholar 

  7. 7

    Ilangumaran, S., Ramanathan, S., La Rose, J., Poussier, P. & Rottapel, R. Suppressor of cytokine signaling 1 regulates IL-15 receptor signaling in CD8+CD44high memory T lymphocytes. J. Immunol. 171, 2435–2445 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Krutzik, P.O., Clutter, M.R. & Nolan, G.P. Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry. J. Immunol. 175, 2357–2365 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Ilangumaran, S., Finan, D. & Rottapel, R. Flow cytometric analysis of cytokine receptor signal transduction. J. Immunol. Methods 278, 221–234 (2003).

    CAS  Article  Google Scholar 

  10. 10

    Kersh, E.N. et al. TCR signal transduction in antigen-specific memory CD8 T cells. J. Immunol. 170, 5455–5463 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Chow, S., Patel, H. & Hedley, D.W. Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 46, 72–78 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Zell, T. et al. Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo. Proc. Natl. Acad. Sci. USA 98, 10805–10810 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Krutzik, P.O., Hale, M.B. & Nolan, G.P. Characterization of the murine immunological signaling network with phosphospecific flow cytometry. J. Immunol. 175, 2366–2373 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Irish, J.M. et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Clutter, M.R., Krutzik, P.O. & Nolan, G.P. Phospho-specific flow cytometry in drug discovery. Drug Discov. Today Technol. 2, 295–302 (2005).

    Article  Google Scholar 

  16. 16

    Jacobberger, J.W. et al. Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity. Cytometry A 54, 75–88 (2003).

    Article  Google Scholar 

  17. 17

    Kuckuck, F.W., Edwards, B.S. & Sklar, L.A. High throughput flow cytometry. Cytometry 44, 83–90 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Edwards, B.S., Oprea, T., Prossnitz, E.R. & Sklar, L.A. Flow cytometry for high-throughput, high-content screening. Curr. Opin. Chem. Biol. 8, 392–398 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 1884–1886 (2003).

    CAS  Article  Google Scholar 

  20. 20

    Fulton, R.J., McDade, R.L., Smith, P.L., Kienker, L.J. & Kettman, J.R., Jr. Advanced multiplexed analysis with the FlowMetrix system. Clin. Chem. 43, 1749–1756 (1997).

    CAS  PubMed  Google Scholar 

  21. 21

    Vignali, D.A. Multiplexed particle-based flow cytometric assays. J. Immunol. Methods 243, 243–255 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Bradford, J.A., Buller, G., Suter, M., Ignatius, M. & Beechem, J.M. Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry. Cytometry A 61, 142–152 (2004).

    Article  Google Scholar 

  23. 23

    Zhang, J.H., Chung, T.D. & Oldenburg, K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    CAS  Article  Google Scholar 

  24. 24

    Nam, S. et al. Indirubin derivatives inhibit Stat3 signaling and induce apoptosis in human cancer cells. Proc. Natl. Acad. Sci. USA 102, 5998–6003 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Lagasse, E. & Weissman, I.L. Flow cytometric identification of murine neutrophils and monocytes. J. Immunol. Methods 197, 139–150 (1996).

    CAS  Article  Google Scholar 

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We thank E. Danna and R. Wolkowicz for critical reading of the manuscript and J. Crane for technical support. P.O.K. was supported by a Howard Hughes Medical Institute predoctoral fellowship. G.P.N. was supported by National Heart Lung and Blood Institute contract N01-HV-28183 and National Institutes of Health grant AI35304.

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Corresponding author

Correspondence to Garry P Nolan.

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Competing interests

G.P.N. and P.O.K. are each paid consultants to Becton Dickenson, a provider of antibodies and flow cytometry–based reagents. G.P.N. consults occasionally with multiple pharmaceutical and biotechnology companies about flow cytometry–based analysis of signaling systems.

Supplementary information

Supplementary Fig. 1

Increasing cell number to antibody ratio does not significantly impact phospho-antibody staining. (PDF 486 kb)

Supplementary Fig. 2

Detailed titration of four hits from Jurkat drug screening experiment. (PDF 284 kb)

Supplementary Methods (PDF 67 kb)

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Krutzik, P., Nolan, G. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 3, 361–368 (2006). https://doi.org/10.1038/nmeth872

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