Intravascular staining for discrimination of vascular and tissue leukocytes

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.

Tissue Distribution of Memory T and B Cells in Rhesus Monkeys following Influenza A Infection

“…Similar to influenza-infected mice (51), influenza infection in rhesus monkeys generates two populations of lung memory T cells, ones expressing T RM cell phenotypic markers CD103 and CD69, which are involved in cell adhesion and tissue retention and likely localize in lung parenchyma, whereas the others lack CD69 and CD103 expression and thus likely localize in lung vasculature. Unlike mice in which the anatomical location of vascular and lung tissue memory T cells can be differentiated by intravascular Ab staining combined with tetramer labeling (51,52), there are significant technical restrictions to using such a technique in rhesus monkeys.…”

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration

“…For homeostatic trafficking in untreated recipient mice, 5 × 10 6 dKO and WT dye-labeled cells were transferred intravenously into recipient mice which were then euthanized 2 or 24 h after adoptive transfer and peripheral lymph nodes, spleen, blood, and lungs were harvested. Intravascular staining of cells remaining in blood vessels was performed via intravenous injection of 3 μg PE-or APCconjugated anti-CD4 antibody (GK1.5) 3 min before euthanasia (39). After euthanizing with CO 2 , blood was then harvested by cardiocentesis, and the peripheral vasculature and lungs were fully perfused through the heart with saline.…”

“…Activated T cells can become lodged in the vasculature (particularly in the lungs) (37,38), potentially because of their increased size and adhesion properties. Therefore, we used an established technique (39,40) to distinguish extravasated T cells from those stuck intravascularly by injecting intravenously a fluorophoreconjugated anti-CD4 antibody immediately before euthanasia of the recipient mice (Fig. 1B).…”

Intranasal Introduction of Fc-Fused Interleukin-7 Provides Long-Lasting Prophylaxis against Lethal Influenza Virus Infection

“…At 7 and 14 days after IL-7-mFc treatment, mice were injected with 2.5 g of antimouse CD3e-peridinin chlorophyll protein (PerCP)-Cy5.5 (BD bioscience) intravenously at 10 min prior to sacrifice as previously described (31). Residual antibody was removed by cardiac perfusion with PBS, and the lung resident T cell populations in single-cell suspensions of lung homogenate were analyzed by flow cytometry.…”