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Intravascular staining for discrimination of vascular and tissue leukocytes

Nature Protocols volume 9, pages 209–222 (2014)Cite this article

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Abstract

Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5–30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses.

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Figure 1: Evidence that perfusion should be avoided.
Figure 2: Technical considerations for intravascular staining.
Figure 3: Intravascular staining is confined to vascular cells.
Figure 4: Intravascular staining without perfusion is sufficient to reveal unique lymphocyte subsets in tissues.
Figure 5: Intravascular staining indicates anatomic localization of B cells.
Figure 6: Intravascular staining during Mtb infection reveals tissue-specific myeloid cell subsets.
Figure 7: Intravascular staining reveals myeloid and lymphoid tissue–specific subsets in a mouse renal adenocarcinoma model.

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Acknowledgements

This study was supported by US NIH grant nos. AI084913-01 and DP2 OD006467 (D.M.), the Arnold and Mabel Beckman Foundation (D.M.), grant no. T90DE022732 from the National Institute of Dental and Craniofacial Research (K.G.A.), the National Institute of Allergy and Infectious Diseases Intramural Program (K.M.-B. and D.L.B.) and US NIH grant no. CA109446 (T.S.G.). We thank the University of Minnesota Center for Immunology Imaging Core, the University of Minnesota Imaging Center and the Flow Cytometry Core.

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  1. Heungsup Sung

    Present address: Present address: Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea.,

Authors and Affiliations

  1. Department of Microbiology, Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA

    Kristin G Anderson, Heungsup Sung, Lalit Beura, Justin J Taylor, Lindor Qunaj, Vaiva Vezys & David Masopust

  2. Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, US National Institutes of Health (NIH), Bethesda, Maryland, USA

    Katrin Mayer-Barber

  3. Department of Urology, Center for Immunology, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

    Britnie R James & Thomas S Griffith

  4. T lymphocyte Biology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA

    Daniel L Barber

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Contributions

K.G.A., K.M.-B., H.S., L.B., B.R.J., J.J.T., T.S.G., V.V., D.L.B. and D.M. designed the experiments. K.G.A., K.M.-B., H.S., L.B., B.R.J., L.Q. and D.L.B. performed the experiments and analyzed data. K.G.A. and D.M. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Imaging intravascular staining of leukocytes in unperfused tissues.

C57Bl/6 mice were injected with anti-CD45.2 mAb i.v. 12d after i.t. LCMV infection. (a,b) Salivary gland, (c,d) female reproductive tract, (e-g) small intestine, and (h,i) large intestine sections showing anti-CD45.2 i.v. mAb staining (red) and ex vivo DAPI (gray), anti-cytokeratin 8 and -cytokeratin 18 (green), and -CD31 (cyan) staining. Scale bars represent 100μm. All data are representative of three experiments from nine mice.

Supplementary Figure 2 Intravascular staining profile of endogenous LCMV-specific CD8 and CD4 T cells isolated from several unperfused tissues.

C57Bl/6 mice were injected with anti-CD45.2 mAb i.v. 12d after i.t. LCMV infection. Lymphocytes were isolated and LCMV-specific CD4+ and CD8+ T cells were identified with I-Ab/gp66-77 and H2-Db/gp33-41 MHC tetramers, respectively. Enumeration of anti-CD45.2 i.v. negative I-Ab/gp66-77 gated CD4 and H2-Db/gp33-41 CD8 T cells isolated from the indicated compartments. * P = 0.05. **** P < 0.0001, based on a two-tailed student's test with a 95% confidence interval. N.D. = not detectable. Error bars indicate SEM.

Supplementary information

Imaging intravascular staining of leukocytes in unperfused tissues.

C57Bl/6 mice were injected with anti-CD45.2 mAb i.v. 12d after i.t. LCMV infection. (a,b) Salivary gland, (c,d) female reproductive tract, (e-g) small intestine, and (h,i) large intestine sections showing anti-CD45.2 i.v. mAb staining (red) and ex vivo DAPI (gray), anti-cytokeratin 8 and -cytokeratin 18 (green), and -CD31 (cyan) staining. Scale bars represent 100μm. All data are representative of three experiments from nine mice. (PDF 804 kb)

Intravascular staining profile of endogenous LCMV-specific CD8 and CD4 T cells isolated from several unperfused tissues

C57Bl/6 mice were injected with anti-CD45.2 mAb i.v. 12d after i.t. LCMV infection. Lymphocytes were isolated and LCMV-specific CD4+ and CD8+ T cells were identified with I-Ab/gp66-77 and H2-Db/gp33-41 MHC tetramers, respectively. Enumeration of anti-CD45.2 i.v. negative I-Ab/gp66-77 gated CD4 and H2-Db/gp33-41 CD8 T cells isolated from the indicated compartments. * P = 0.05. **** P < 0.0001, based on a two-tailed student's test with a 95% confidence interval. N.D. = not detectable. Error bars indicate SEM. (PDF 322 kb)

mAbs injected i.v. are restricted to capillary vasculature in the lung, Example 1.

Anti-CD31 and anti-CD45.2 mAb were injected i.v. into P14 chimeric mice 12d after i.t. LCMV infection. Confocal imaging of lung with anti-CD31 i.v. (green), -CD45.2 i.v. (red), and ex vivo anti-CD8β (blue) staining. Representative of three experiments totaling 4 mice. (AVI 40902 kb)

mAbs injected i.v. are restricted to capillary vasculature in the lung, Example 2.

Anti-CD31 and anti-CD45.2 mAb were injected i.v. into P14 chimeric mice 12d after i.t. LCMV infection. Confocal imaging of lung with anti-CD31 i.v. (green), -CD45.2 i.v. (red), and ex vivo anti-CD8β (blue) staining. Representative of three experiments totaling 4 mice. (AVI 38616 kb)

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Anderson, K., Mayer-Barber, K., Sung, H. et al. Intravascular staining for discrimination of vascular and tissue leukocytes. Nat Protoc 9, 209–222 (2014). https://doi.org/10.1038/nprot.2014.005

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