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Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry

Nature Protocols volume 5, pages 357–370 (2010)Cite this article

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Abstract

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (APCs; e.g., dendritic cells, monocytes and B lymphocytes) in minimally manipulated whole blood samples. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, in order to show the quantification of surface expression levels of molecules involved in chemotaxis (e.g., CX3CR1 and CCR2), adhesion (e.g., CD11b and CD62L), antigen presentation (e.g., CD83, CD86 and CD209) and immune regulation (e.g., CD101) on circulating APCs. Each immunostaining reaction requires as little as 50–100 μl of peripheral whole blood and no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h.

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Figure 1: Diagram illustrating the workflow for multiplexed immunophenotypic analysis of the major circulating antigen-presenting cell subsets by flow cytometry.
Figure 2: Flow cytometric analysis of monocyte subsets and CD19+HLA-DR+ B lymphocytes in whole blood.
Figure 3: A time-course experiment showing the natural differentiation or transition of CD14+ monocytes into CD14+CD16+ cells (pink arrow at 24 h) in citrated whole blood sample even in the absence of any stimulus.
Figure 4: The systematic display of antibody–fluorochrome pairs created in the worksheet window of the BD FACSDiva software is useful in organizing large number of bivariate plots for visualizing the correct spectral overlap compensation, quality control and for discovering novel cell subsets.
Figure 5: Analysis of two major DC subsets in whole blood.
Figure 6: DC subset-defining markers for identifying MDCs and PDCs as described in Protocol B.

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Acknowledgements

We thank the Wellcome Trust, the Juvenile Diabetes Research Foundation (JDRF) International and the National Institute for Health Research Cambridge Biomedical Research Centre for funding. The Cambridge Institute for Medical Research is a recipient of the Wellcome Trust Strategic Award (079895). E.F. was funded by the JDRF and the Prince Philip Graduate Exhibition from the Cambridge Overseas Trust. L.S.W. is a Wellcome Trust Principal Research Fellow.

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Authors and Affiliations

  1. Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK

    Erik Fung, Laura Esposito, John A Todd & Linda S Wicker

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  1. Erik Fung
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Contributions

E.F. contributed to the conception, design, performance and analysis of the protocols and wrote the manuscript. L.E. contributed to the configuration and establishment of the flow cytometer in the laboratory and to the writing of the manuscript. J.A.T. participated in the conception of the protocols and the writing of the manuscript. L.S.W. contributed to the conception, design and analysis of the protocols and to the writing of the manuscript.

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Correspondence to Linda S Wicker.

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Fung, E., Esposito, L., Todd, J. et al. Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry. Nat Protoc 5, 357–370 (2010). https://doi.org/10.1038/nprot.2009.246

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