OMIP‐104: A 30‐color spectral flow cytometry panel for comprehensive analysis of immune cell composition and macrophage subsets in mouse metabolic organs

Abstract Obesity‐induced chronic low‐grade inflammation, also known as metaflammation, results from alterations of the immune response in metabolic organs and contributes to the development of fatty liver diseases and type 2 diabetes. The diversity of tissue‐resident leukocytes involved in these metabolic dysfunctions warrants an in‐depth immunophenotyping in order to elucidate disease etiology. Here, we present a 30‐color, full spectrum flow cytometry panel, designed to (i) identify the major innate and adaptive immune cell subsets in murine liver and white adipose tissues and (ii) discriminate various tissue‐specific myeloid subsets known to contribute to the development of metabolic dysfunctions. This panel notably allows for distinguishing embryonically‐derived liver‐resident Kupffer cells from newly recruited monocyte‐derived macrophages and KCs. Furthermore, several adipose tissue macrophage (ATM) subsets, including perivascular macrophages, lipid‐associated macrophages, and pro‐inflammatory CD11c + ATMs, can also be identified. Finally, the panel includes cell‐surface markers that have been associated with metabolic activation of different macrophage and dendritic cell subsets. Altogether, our spectral flow cytometry panel allows for an extensive immunophenotyping of murine metabolic tissues, with a particular focus on metabolically‐relevant myeloid cell subsets, and can easily be adjusted to include various new markers if needed.