Immunology is a rapidly evolving field of research with sophisticated models and methods. However, detailed data on total immune cell counts and population distributions remain surprisingly scarce. Nevertheless, recently established quantitative approaches could help us understand the overall complexity of the immune system. Here, we studied a major histocompatibility complexclass II - enhanced green fluorescent protein knock-in mouse model to precisely identify and manipulate lymphoid structures. By combining flow cytometry with light sheet microscopy, we quantified MHC II+ populations of the small intestine and associated individual mesenteric lymph nodes, with 36.7 × 106 cells in lamina propria, 3.0 × 105 cells in scattered lymphoid tissue and 1.1 × 106 cells in Peyer's patches. In addition to these whole-organ cell counts, we assessed approximately 1 × 106 total villi in the small intestine and 450 scattered lymphoid tissue follicles. By direct noninvasive microscopic observation of a naturally fully translucent mouse organ, the cornea, we quantified 12 ± 4 and 35 ± 7 cells/mm2 Langerhans- and macrophage-like populations, respectively. Ultimately, our findings show that flow cytometry with quantitative imaging data analysis enables us to avoid methodological discrepancies while gaining new insights into the relevance of organ-specific quantitative approaches for immunology.

Center for Translational Immunology University Medical Center Utrecht Utrecht the Netherlands

Laboratory of Cell Immunology Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

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