Figure 3. Monocytes/macrophages in the mouse spleen are readily identified without using the F4/80 antigen.

A) Debris, doublets, and dead cells were excluded from total C57BL/6 mouse splenocytes as in Figures 1-2. CD11c^Hi cells were excluded from the CD11b⁺ population, followed by gating into F4/80⁺Ly6G^- and F4/80^-Ly6G^Hi (A) subpopulations. F4/80⁺Ly6G^- cells were divided further into subpopulations B, C, and D based on Ly6C versus SSC. Populations A-D were then isolated by FACS. Cytospin preparations from the sorted cells were stained with Diff-Quick and Giemsa. Subpopulation A (F4/80^-Ly6G^Hi) contained solely neutrophils. Subpopulation B (Ly6C^Lo-negSSC^Hi) contained eosinophils while subpopulations C (Ly6C^Lo-negSSC^Lo) and D (Ly6C⁺SSC^Lo) contained monocytes/macrophages. B) Cells were prepared and gated as in Figure 3A except that NK1.1 was substituted for F4/80 staining to distinguish NK1.1^-Ly6G^- and NK1.1^-Ly6G^Hi (A) subpopulations. NK1.1^-Ly6G^- cells were divided into subpopulations B-D based on Ly6C versus SSC. Subpopulations A-D were then sorted and cytospin preparations were prepared as above. Subpopulation A (Ly6G^HiNK1.1^-) contained exclusively neutrophils. Subpopulation B (Ly6C^Lo-negSSC^Hi) contained eosinophils while subpopulations C (Ly6C^Lo-negSSC^Lo) and D (Ly6C⁺SSC^Lo) contained monocytes/macrophages. For all analyses, frequencies of cells in each sub-gate (after debris and doublet exclusion) are expressed as a percentage of live cells.