Figure 3. A485 mobilizes leukocytes from the bone marrow.

(A) Experimental design (B) Exemplary flow cytometry plot of adoptively transferred CD45.1 neutrophils in the blood and bone marrow 8h post i.v. injection (C) Quantification of endogenous CD45.2 and transferred CD45.1 neutrophils in different organs 5h after A485 injection expressed as percentage of the mean neutrophil number in the respective organ of vehicle treated mice (n=4/group) (D) Estimated loss of transferred CD45.1 neutrophils in the bone marrow vs. gain in the blood (E) Experimental design of intravascular labeling of leukocytes using fluorophore conjugated anti-CD45 antibodies (left panel) with the corresponding gating strategy (right panel). Exemplary plots are shown (F) Extravascular (referred to as “single CD45+”) and intravascular (“double CD45+”) neutrophils in lung tissue 5h after A485 or vehicle injection. Results were normalized according to tissue weight (n=5/group) (G) Intra- or extravascular neutrophils in the bone marrow of the same animals (H) Quantification of leukocytes in the bone marrow and leukocytes and neutrophils in the blood 3 days after sub-lethal irradiation (4Gy) followed by injection of A485 or vehicle (n=3–4/group for bone marrow and n=7/group for blood). Data is shown as mean ± s.e.m.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (C,F,G) unpaired, two-tailed student’s t-test (H) one way ANOVA with Holm-Sidak’s post hoc test.