Characterization of Mφs and monocytes in spleens of naive and 2 week posttransplant MacGreen mice. (A) Representative flow cytometry analysis (n = 5-13) of naive (no-Tx) and 2 week posttransplant MacGreen splenocytes demonstrating extended characterization of gated Mφ and monocyte populations as summarized in Table 1. Specifically, monocyte-Mφs were first enriched by gating F4/80⁺Ly6G^neg cells (gate i) and further subdivided into VCAM-1⁺GFP⁺ naive/recipient Mφs (gate iii and ix), VCAM-1^negGFP⁺ naive/recipient monocytes (gate iv or x), VCAM-1⁺GFP^neg donor Mφs (gate vii), and VCAM-1^negGFP^neg donor monocytes (gate viii). Granulocytes were gated as F4/80^negLy6G⁺ cells (gate ii or vi). B and C) Representative histograms for MerTK (B) and CD169 (C) for all populations of interest as indicated by histogram titles. Antibody expression is shown as red lines, and appropriate isotype staining is shown as blue lines. (D) Number of recipient (blue bars) and donor (white bars) granulocytes per spleen across the transplantation time course. (E) Total number of recipient (dark green bars) and donor (brown bars) Mφ, recipient (light green bars) and donor (yellow bars) monocytes per spleen across the transplantation time course. Data are presented as mean ± SD. Statistical analysis of recipient Mφs was performed using 1-way ANOVA Tukey’s multiple comparison test. In panel E, asterisks and crosshatches indicate significant differences in recipient Mφ number compared with 2 and 3 weeks posttransplant, respectively (^##P < .01 and ****/^####P < .0001). Data represent 5 to 13 samples per time point. Flow cytometry plots and histograms as well as associated gated cell percent frequencies are from a representative animal, and the percentage of positive cells is based on the preceding parent populations. Data are representative of 5 to 13 individual animal samples per time point from either 1 (weeks 3 and 32), 2 (week 5), or 3 (all other time points) biological replicate experiments. 300 000 events were collected from each experiment sample by flow cytometry.