OMIP‐095: 40‐Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues

Abstract High‐dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40‐color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co‐stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre‐clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40‐color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.