Analysis of G2B-10A and R2-T1AS conditioned media. (A) Cytokine antibody array analysis of medium conditioned by G2B-10A cells, plated at a density of 1.5 × 106 cells/ml. (B) Cytokine antibody array analysis of medium conditioned by R2-T1AS cells, plated at a density of 1.875 × 105 cells/ml. The R2-T1AS cells were plated at 8 times lower density to reflect the 1:8 ratio at which cancer and benign cells were co-cultured in the soft agar assays. Cells were cultured in suspension, in low-glucose, 5% serum medium. Conditioned media were harvested after 3 days of incubation and analyzed by cytokine antibody array. Both membranes were incubated with 1 ml of conditioned media derived from the indicated cell lines, underwent the same treatment and were exposed to the X-ray film for the same time. POS - positive control spots, solid lines - cytokines expressed differentially in the two cultures, dashed lines - not reproducible signals. (C) Quantification of the arrays in Figures 6A and 6B. The signals on the G2B-10A array (green bars) and R2-T1AS array (red bars) were quantified by densitometry and normalized to the average of positive control signals (POS). (D) Analysis of MCP-1 concentration in media conditioned by R2-T1AS cells or G2B-10A cells (green bar) by ELISA. nd - not detectable.