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Cytokines and chemokines are responsible for regulating inflammation and the immune response. Cytokine and chemokine release is typically quantitated by ELISA or Western blot analysis. In order to expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based ExcelArray antibody sandwich microarrays. Each slide consists of 16 subarrays (wells) printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the “sandwich” ELISA, in which an analyte protein is “sandwiched” between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked DyLight 649 dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. The Jurkat cell line was used as a model for human T cell leukemia and the A549 cell line was used as model for human non-small cell lung cancer in our experiments. In order to evoke a cytokine/chemokine response, cells were stimulated with Tumor Necrosis Factor alpha (TNFα), Phorbol-12-myristate-13-acetate (PMA, TPA) and phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angiogenesis and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-α, and MIP-1α, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell culture supernatants for the profiling of cellular inflammatory mediator release.