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Figure S4. IL-8 stimulates the migration of monocytes. (A) The migration of monocytes purified from human peripheral blood was assayed in response to either increasing concentrations of purified IL-8 or conditioned medium of SEMA3B and control (EV) tumor cells (10×, as in Fig. 4 B), which was added to the lower chamber of Transwell inserts. Porous membranes had been coated with 10 µg/ml fibronectin. Migrated monocytes were stained with Giemsa and counted as in Fig. 4 B. Purified IL-8 induced monocyte chemotaxis in a dose-dependent manner, in a comparable range as SEMA3B-concentrated medium (estimated to contain 60 ng/ml IL-8; see legend to Fig. 5 C). (B) The chemotactic activity of 100 ng/ml of purified IL-8 on human monocytes, purified as in A, was tested in the presence or absence of 0.5–1 µg/ml IL-8 neutralizing antibody (MAB208) or the same concentration of an unrelated control antibody (directed against CD19, an antigen only expressed by B lymphocytes). Transmigrated monocytes were counted upon Giemsa staining. Values were normalized to control and represent the mean ± SD of two independent experiments. **, P < 0.01; *, P < 0.05. n.t., nontreated cells.