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.
Bibliographical noteFunding Information:
acknoWleDGMents 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.