G-CSF–induced mobilization triggers C3 activation in BM. Mice were mobilized with G-CSF, and BM cells were isolated and examined for bound C3 by staining with affinity-purified antimouse C3–Oregon Green and flow cytometry. In all tests, wild type C3^+/+ (solid lines) were tested in parallel with C3-deficient (C3^–/–) mice (dashed lines). (A) Upper histogram: detection of bound C3 on the entire BM cell population after 3 days of treatment with G-CSF. Middle histogram: examination of CD45⁺ cells (leukocytes) shows that the majority of cells express detectable C3 when compared with the CD45⁺ cells from a C3^–/– mouse (control for antibody specificity). Bottom histogram: analysis of the CD45^– stromal cells shows far more bound C3 is present on stromal cells than on the CD45⁺ leukocytes. (B) Isotype controls with the use of Oregon Green–conjugated nonspecific IgG. (C) For each test, BM cells from mice that were either not treated or treated with G-CSF were examined in parallel to show that G-CSF treatment selectively produced C3 staining with wild-type but not with C3-deficient mice. Left panel: detection of bound C3 on the entire BM cell population from a C3^–/– mouse (KO) after 3 days of treatment with G-CSF (solid line) or nontreated animals (dotted line). Right panel: detection of bound C3 on the entire BM cell population from a wild-type mouse (WT) after 3 days of treatment with G-CSF (solid line) or nontreated animals (dotted line). An additional control for the lack of dead-cell nonspecific staining used a nonspecific Oregon Green–labeled IgG Ab that exhibited the same low level of staining with both the wt and C3-deficient mouse BM cells (not shown). This experiment was repeated 3 times with similar results.