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

Abstract

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.

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Acknowledgements

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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Correspondence to Garry P Nolan.

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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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