immune cells resident in adipose tissue have important functions in local and systemic metabolic homeostasis. Nevertheless, these immune cell populations remain poorly characterized in bovines. Recently, we described diverse lymphocyte subpopulations in adipose tissue of Holstein-Friesian cows. Here, we aimed at characterising myeloid cell populations present in bovine adipose tissue using multicolour flow cytometry, cell sorting and histochemistry/immunohistochemistry. Macrophages, CD14 + CD11b + MHc-ii + CD45 + cells, were identified in mesenteric and subcutaneous adipose tissue, though at higher proportions in the latter. Mast cells, identified as SSC-A high CD11b −/+ CD14 − MHcii − CH138A − CD45 + cells, were also observed in adipose tissue and found at higher proportions than macrophages in mesenteric adipose tissue. Neutrophils, presenting a CH138A + CD11b + phenotype, were also detected in mesenteric and subcutaneous adipose tissue, however, at much lower frequencies than in the blood. Our gating strategy allowed identification of eosinophils in blood but not in adipose tissue although being detected by morphological analysis at low frequencies in some animals. A population not expressing CD45 and with the CH138A + CD11b − MHc-ii − phenotype, was found abundant and present at higher proportions in mesenteric than subcutaneous adipose tissue. The work reported here may be useful for further studies addressing the function of the described cells.www.nature.com/scientificreports www.nature.com/scientificreports/ colour-flow cytometry revealed the presence of CD14 + cells, CD172a + cells, CD11c + cells and CD163 + cells in the stromal vascular fraction (SVF) of subcutaneous and omental adipose tissue 23,24 . Recently, Aylward et al. 25 reported the presence of CD172a + CD11b + cells, CD11c + MHC-II + cells, and CD11b + CD11c + cells in mesenteric adipose tissue. However, these markers are not exclusive to macrophages but common to other myeloid cell populations or can be even expressed in non-hematopoietic cells. As an example, studies in humans and mice have shown that CD14 can be expressed in non-myeloid cells 26 such as preadipocytes 27 and epithelial cells 28 . Also, CD172a is expressed by granulocytes such as eosinophils 29 and mast cells 30 beside its expression in monocytes/ macrophages. Since mast cells 31,32 and eosinophils 3,33 have already been described in adipose tissue, single markers analysis limits the interpretation of the data. Therefore, to characterize the different myeloid cell populations in bovine adipose tissue we used a multicolour flow cytometry panel associated with cell sorting and histochemistry/immunohistochemistry analysis. Our strategy allowed identification and isolation of macrophages, mast cells and neutrophils with macrophages being observed at different proportions in mesenteric adipose tissue (MAT) and subcutaneous adipose tissue (SAT). We also showed that CD45 negative cells represent more than 40% of all the cells in the adipose tissue SVF of MAT. A non-leukocyte populat...