Fig. 5. Supernatants of U251MG-Nef_Bru cells do not induce chemotaxis of CCR2-deficient monocytic cells. (A) Immunocytochemistry analysis of monocytic THP-1 and U-937 cells using anti-CCR2 Ab. To exclude non-specific staining by the secondary antibody, anti-CCR2 Ab was substituted with distilled water (Control). Images were obtained using a microscope (Axiolab, Carl-Zeiss Jena) equipped with a x40 objective and a x10 ocular lenses. Original magnification, x100. (B) CCR2-specific RT-PCR with total RNA originating from THP-1 cells (T) and U-937 cells (U). GAPDH served as internal control. (C) Supernatants of U251MG-Nef_Bru-4/4.2, -parental, -pNeo cells and rCXCL12/SDF-1 (25ng/ml) were placed into the lower parts of the chemotaxis unit, and U-937 cells in the upper parts. One representative experiment is shown. Data represent mean ± s.e.m. of triplicates.