Quantitative measurement of proteins may be accomplished with multiplex micro-array technology. Nitrocellulose membranes affixed to glass slides (NM-Slides) have been used for spotting the capture antibodies with detection by Cy5. While this system works well for serum and tissue culture medium, organ homogenates substantially interfere with the assay due to high backgrounds and "flattened" standard curves. We investigated whether IR dyes, which are not affected by autoflourescence, would result in improved standard curves. A total of 21 capture antibodies (350-400 picoliters/spot) were spotted on either NMSlides or 96-well ELISA plates using a non-contact (Piezorray) spotting system. Both systems were incubated with blocking buffer followed by overnight incubation with cytokine standards. Two sets of standards were prepared, one composed of buffer containing organ homogenates and the other in buffer alone. After washing, slides or plates were incubated with biotinconjugated anti-cytokine antibodies, followed by incubation with Cy5 conjugated streptavidin (for NM-Slides) or streptavidin IRDye 800 (for Infrared imaging system). NM-Slides were read using an Axon Genepix 4000 system and the plate read using the Odyssey IR system. The detection limit and dynamic range obtained from infrared imaging system was significantly lower than the data from Genepix data acquisition system. When we compared the NM-Slide with the 96-well plate using the IR dyes, the latter provide a better lower limit of detection and wider dynamic ranges. In conclusion, for multiplex analysis of cytokines and chemokines in protein rich matrixes, such as organ homogenates, the 96-well plate with infrared imaging system prove to be the best method for detection.Figure