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Disk sorter ultimate 9.7.141/7/2023 Although these markers primarily identify lymphoid cells, some lymphoid cells are known to express low levels of CD11b, CD11c, and SiglecF, particularly when activated, which could lead to misclassification of cell types. After gating on CD45 + cells, lymphoid cells were identified by using a “dump” gate consisting of a mixture of antibodies specific for Thy1, CD19, and NK1.1 (a). In addition to eliminating debris, doublets, and dead cells from our analysis, an essential part of our strategy was to exclude lymphoid cells. S2 Fig: A “Dump” strategy excludes lymphoid cells. In the Mtb infected lung, siglecF -CD11c HiCD11b Hi cells are likely to be a mixture of CD11bDC and CD11c Hi MDC. Finally, the remaining CD11c Hi cells were designated as DC, and 2 different subsets identified by their expression of CD103 (i.e., CD103DC or cDC1, “6”) or CD11b (i.e., CD11bDC or cDC2, “7”). CD11c intCD11b Hi cells were identified as recruited macrophages (RM, “5”) and monocytes, and monocytes were further distinguished by their Ly6C expression (“4”). AM (“2”) were identified by their siglecF HiCD11c Hi phenotype, while eosinophils (Eos, “3”) expressed lower amounts of SiglecF and no CD11c (i.e., siglecF intCD11c -) (g). Neutrophils (PMNs, “1”) were identified based on Ly6g expression (f). Leukocytes in the lung were identified based on CD45 staining (d) and lymphoid cells were excluded by using a dump channel that consisted of anti-Thy1, CD19, and NK1.1 (e). Single cell suspensions obtained from the lungs of mice were gated based on (a) scatter, (b) singlets, and then dead cells excluded (c). Therefore, the following scheme takes advantage of SiglecF to identify AM, independently of MHCII. However, during inflammation, AM upregulate both CD11c and MHCII. This strategy works well for uninfected mice housed under conventional conditions. CD11b and CD11c are frequently used to distinguish alveolar macrophages (AM) from pulmonary dendritic cells (DC): AM are generally CD11b loCD11c hiMHCII low-int, while DC are CD11b int-hiCD11c hiMHCII hi. S1 Fig: Identification of 7 populations of myeloid cells in the lungs of Mtb-infected mice.
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