Purification of human and mouse monocyte subsets. (A) Dot plot on the left shows staining of PBMCs for human monocytes before isolation of CD16⁻ and CD16⁺ subsets. Middle and right dot plots show representative samples of isolated human monocyte subsets, revealing purities of 93% or greater. The purified CD14⁺CD16⁺⁺ cells contained 7.5% intermediate monocytes in this example. (B) In mouse whole blood leukocyte preparations, SSC-H voltage was set to exclude a majority of granulocytes and low SSC-H cells were gated (first dot plot). B220⁺ cells were excluded (second dot plot) and subsequent gating on CD115⁺ F4/80⁺ cells selectively identified monocytes (third dot plot). Ly-6C⁺ and Ly-6C^lo monocyte subsets were identified by staining with anti–Gr-1 antibody, which recognizes both Ly-6G and Ly-6C (last dot plot). Only Ly-6C is expressed on monocytes. Ly-6C⁺/Gr-1⁺ and Ly-6C^lo/Gr-1^lo monocyte subset postsort purity was 95% to 97% (not shown).