(A) Schematic representation of experimental design. Transwell culture of purified BM Ly-6C^hiMHC-II^– CMo placed above mouse endothelial cells (ECs) seeded in the upper compartment for 2 days. Monocytes in the bottom compartment are considered as having transmigrated through the ECs, while the ones remaining in the top well are considered the nonmigrated subpopulation. (B) Representative histograms showing the gating strategy for Ly-6C^hiMHC-II^– CMo, Ly-6C^lo/–MHC-II^– PMo, and Ly-6C⁺MHC-II⁺ transient macrophages in the Transwell culture, as shown in A. Numbers represent the frequency of Ly-6C^hiMHC-II^– CMo (98.8, 46.1, and 9.17 before culture, nonmigrated cells, and migrated cells, respectively), Ly-6C^lo/–MHC-II^– PMo (0.68, 47.5, and 1.15 before culture, nonmigrated cells, and migrated cells, respectively), and Ly-6C⁺MHC-II⁺ transient macrophages (0.12, 2.67, and 89.2 before culture, nonmigrated cells, and migrated cells, respectively). (C) Bar graph showing expression of surface markers on cultured monocytes, as shown in A (n = 6). (D) Bar graph showing the monocyte numbers in cocultures of purified BM Ly-6C^hiMHC-II^– CMo layered above mouse ECs, which had been pretreated with blocking antibody against P selectin, VCAM-1, ICAM-1, E selectin, CD11b, or isotype control (10 ng/mL, n = 6). (E) Bar graph showing absolute number of circulating Ly-6C^hi CMo and Ly-6C^lo/– PMo at 20 hours in hemin-injected (35 μmol/kg body weight) WT mice pretreated for 30 minutes with anti–P selectin blocking antibody (5 mg/kg body weight, i.v.), anti–VCAM-1 blocking antibody (5 mg/kg body weight, i.v), or isotype control antibody (5 mg/kg body weight, i.v) (n = 6). (F) Bar graph showing absolute numbers of liver Ly-6C^hiMHC-II^– CMo and Ly-6C⁺MHC-II⁺ transient macrophages (tMΦ) in mice, injected as in D (n = 6). Each symbol represents data from an individual mouse. Data are shown as the mean ± SEM and were compared using 2-way ANOVA with Bonferroni’s multiple comparisons. *P < 0.05.