Macrophage subsets in WT mice at steady state and during response to stress. (A) Morphology and expression of F4/80, CD11b, and VCAM-1 was analyzed in EI preparations isolated from spleens of PHZ-treated or Epo-treated mice on day 4; Hema3 stain: note the different maturation stages of Ebs that adhere to themselves and to the central macrophage engorged in damaged red cells after PHZ treatment (i); and DAPI-counterstained EIs (ii-v). Light green stain for F4/80 and magenta nuclear stain (assigned color) for surrounding Ebs (ii); light green CD11b⁺ cells (iii); dotted circles indicate central macrophages. Light green F4/80⁺ cells and DAPI-stained Ebs (iv); and green VCAM-1 positivity on central macrophages (v). CD169 expression on F4/80⁺ cells was analyzed by FACS after EI disaggregation (bottom right panel). (B) Percentage of F4/80⁺ cells in the BM and spleen that are CD11b^hi or CD11b^lo at steady state (CD11b^hi: red; CD11b^lo: blue). VCAM-1 expression in the 2 subsets (right panel). Note that only CD11b^lo express VCAM-1 in the spleen. (C) Quantitative kinetic changes in the 2 F4/80⁺ subsets after PHZ or Epo challenge. (D) Quantitative changes in the 2 F4/80⁺ subsets in transplanted mice (WT→WT) treated with PHZ 9 weeks after transplantation (left). Cycling status (Ki-67 antibodies; see “Methods”) of the 2 subsets before and after stress (right). Note differences only in the CD11b^lo subset. The high Ki-67 positivity in this subset changed from 9.63 ± 1.27% to 45.9 ± 6.15% and finally to 76.15 ± 1.25% on days 0 (light blue), 4 (peach) and 6 (red), respectively. No such population was present in CD11b^hi subset (arrow). Number of mice: day 0, n = 6; PHZ-induced stress, day 4, n = 4; day 6, n = 6; and Epo-induced stress, n = 3. Images in (Ai,iv-v) were taken with a Leica DMLB camera (objective N PLAN 40×/0.65, eye piece HC PLAN 10×/22) and 20× objective for (Aii-iii). d, day; DAPI, 4,6 diamidino-2-phenylindole; Spl, spleen.