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Mass cytometry profiling of human dendritic cells in blood and tissues

Nature Protocols volume 16, pages 4855–4877 (2021)Cite this article

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Abstract

The immune system comprises distinct functionally specialized cell populations, which can be characterized in depth by mass cytometry protein profiling. Unfortunately, the low-throughput nature of mass cytometry has made it challenging to analyze minor cell populations. This is the case for dendritic cells, which represent 0.2–2% of all immune cells in tissues and yet perform the critical task of initiating and modulating immune responses. Here, we provide an optimized step-by-step protocol for the characterization of well-known and emerging human dendritic cell populations in blood and tissues using mass cytometry. We provide detailed instructions for the generation of single-cell suspensions, sample enrichment, staining, acquisition and data analysis. We also include a barcoding option that reduces acquisition variability and allows the analysis of low numbers of dendritic cells, i.e., ~20,000. In contrast to other protocols, we emphasize the use of negative selection approaches to enrich for minor populations of immune cells while avoiding their activation. The entire procedure can be completed in 2–3 d and can be conveniently paused at several stages. The procedure described in this robust and reliable protocol allows the analysis of human dendritic cells in health and disease and during vaccination.

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Fig. 1: Schematic representation of the experimental procedures described in this protocol.
Fig. 2: Enrichment of myeloid cells using negative selection.
Fig. 3: Gating of cells to de-barcode.
Fig. 4: Gating of sorted DCs pooled with mouse filler splenocytes.
Fig. 5: Gating of DCs for unbiased analysis.
Fig. 6: Anticipated results for the analysis of DCs from blood, spleen and skin.

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Data availability

Data in this manuscript are available at http://flowrepository.org/ (accession number FR-FCM-Z453), including data shown in Figs. 4–6 and Supplementary Fig. 1. PBMC staining using CD88/CD89 is available upon request to the corresponding author.

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Acknowledgements

This work was supported by NIH grants DP2AR069953, R01CA219994, R01AI158808 and R21AI163775 awarded to J.I. Data collection was performed on an instrument in the Shared FACS Facility obtained using NIH S10 Shared Instrument Grant S10OD016318-01. We thank the blood donors for their participation and the Idoyaga Lab members for technical support and discussions.

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Authors and Affiliations

  1. Microbiology & Immunology Department and Immunology Program, Stanford University School of Medicine, Stanford, CA, USA

    Marcela Alcántara-Hernández & Juliana Idoyaga

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  1. Marcela Alcántara-Hernández
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  2. Juliana Idoyaga
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Contributions

M.A-H. and J.I. conceptualized and wrote the paper, M.A-H. performed the experiments and analyzed the data. J.I. is responsible for funding acquisition, supervision and project administration.

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Correspondence to Juliana Idoyaga.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Gustavo Menezes and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Alcántara-Hernández, M. et al. Immunity 47, 1037–1050.e6 (2017): https://doi.org/10.1016/j.immuni.2017.11.001

Leylek, R. et al. Cell Rep. 32, 108180 (2020): https://doi.org/10.1016/j.celrep.2020.108180

Leylek, R. et al. Cell Rep. 29, 3736–3750.e8 (2019): https://doi.org/10.1016/j.celrep.2019.11.042

Extended data

Extended Data Fig. 1 Frequencies of leukocytes in blood and skin samples before and after enrichment.

a, PBMCs were obtained from blood (Steps 1–10). b, Skin cell suspensions were obtained after enzymatic digestion (Steps 23–37). Myeloid cells or leukocytes were negatively enriched from blood and skin, respectively, following the procedures described in Steps 45–53. Cells were stained for flow cytometry before and after negative enrichment. One representative blood and skin sample is shown. Numbers represent frequency in the gate. Antibodies used are as follows: CD45 Brilliant Violet 785 (Biolegend, cat. no. 304049), CD3 PerCPCy5.5 (Biolegend, cat. no. 300430), CD19 PerCPCy5.5 (Biolegend, cat. no. 302230), CD335 PerCPCy5.5 (Biolegend, cat. no. 331920), CD66b PerCPCy5.5 (Biolegend, cat. no. 305108). Figure created for this protocol from data obtained as part of a primary publication5.

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Alcántara-Hernández, M., Idoyaga, J. Mass cytometry profiling of human dendritic cells in blood and tissues. Nat Protoc 16, 4855–4877 (2021). https://doi.org/10.1038/s41596-021-00599-x

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