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Nat Protoc. 2014 Jan;9(1):209-22. doi: 10.1038/nprot.2014.005. Epub 2014 Jan 2.

Intravascular staining for discrimination of vascular and tissue leukocytes.

Author information

1
Department of Microbiology, Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA.
2
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, US National Institutes of Health (NIH), Bethesda, Maryland, USA.
3
1] Department of Microbiology, Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA. [2].
4
Department of Urology, Center for Immunology, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.
5
T lymphocyte Biology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA.

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.

PMID:
24385150
PMCID:
PMC4428344
DOI:
10.1038/nprot.2014.005
[Indexed for MEDLINE]
Free PMC Article

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