Figure 1. Gr-1 and Ly6C/Ly6G markers identify similar populations of monocytes and macrophages in the mouse spleen.

Total mouse splenocytes from C57BL/6 mice were prepared as described in Materials and Methods. Debris (SSC-A vs. FSC-A) and doublets (FSC-H vs. FSC-A) were excluded and live/dead discrimination was determined using the amine reactive dye Aqua (FSC-H vs. Pacific Orange-A Live/Dead). CD11b⁺Lineage^- cells (gated out using a CD4, CD8, CD19 PerCP-Cy5.5 dump channel) were then sub-gated on CD11b⁺NK1.1^- cells, followed by excluding CD11c^Hi cells. For the first cocktail (CK1), 4 subpopulations were identified: A) Gr-1^HiSSC^Int cells (neutrophils) and B) Gr-1^Lo-negSSC^Hi cells (eosinophils) were negative for MHC Class II and CD115 staining (Supplemental Figure 5) C) Gr-1^Lo-negSSC^Lo cells (monocytes/macrophages) were MHC Class II^+/-CD115^- (red histogram) D) Gr-1⁺SSC^Lo cells (monocytes/macrophages) were MHC Class II^-CD115^+/- (blue histogram). For the second cocktail (CK2), 4 subpopulations were identified: A) Ly6G^HiCD11b⁺ cells (neutrophils) and B) Ly6C^Lo-negLy6G^-SSC^Hi cells (eosinophils) were negative for MHC Class II and CD115 (Supplemental Figure 5) C) Ly6C^Lo-negLy6G^-SSC^Lo cells (monocytes/macrophages) were MHCII^+/-CD115^- (red histogram) D) Ly6C⁺Ly6G^-SSC^Lo cells (monocytes/macrophages) were MHC Class II^-CD115^+/- (blue histogram). Frequencies of cells in each sub-gate (after debris and doublet exclusion) are expressed as a percentage of live cells.