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Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry

Abstract

Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses.

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Figure 1: Technical aspects of quantum dot detection.
Figure 2: Materials used to manufacture the outer layer of quantum dots have a significant impact on level of non-specific staining.
Figure 3: Relative brightness of quantum dots.
Figure 4: Seventeen-color analysis of antigen-specific T cells using quantum dots.

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Acknowledgements

We are grateful to the members of the Laboratory of Immunology at the Vaccine Research Center for their continuing enthusiasm, support and critical suggestions during the development of these technologies. We gratefully acknowledge the assistance of L. Duckett (BD Biosciences) and P. Millman (Chroma Technology Corp.) in optimizing the instrumentation and optics for detection of the quantum dot reagents. D. Price is a Medical Research Council (MRC) Clinician Scientist. Quantum dot materials were developed with partial support from the US National Institutes of Health/National Institute of Biomedical Imaging and BioEngineering Research Partnership program (R01 EB 000364) and the National Institute of Standards and Technology Advanced Technology Program (70NANB0H3000). This work is supported by the Intramural Research Program of the US National Institutes of Health, Vaccine Research Center, National Institute of Allergy and Infectious Diseases.

Author information

Authors and Affiliations

Authors

Contributions

P.K.C. designed and performed experiments, and wrote the manuscript. M.P.B. and M.R. designed experiments. D.A.P., T.F.H., M.R.B., J.Y., E.G., S.P.P., P.G., R.A.K., and S.C.D. provided instrumentation, reagent manufacture and validation, and sample support. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Mario Roederer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1 (PDF 256 kb)

Supplementary Table 1

Summary of phenotypes of antigen-specific CD8 T cells. (PDF 58 kb)

Supplementary Table 2

Reagents used in 17-color staining panel. (PDF 58 kb)

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Chattopadhyay, P., Price, D., Harper, T. et al. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12, 972–977 (2006). https://doi.org/10.1038/nm1371

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