Multiplexed Analysis of Human Cytokines by Use of the FlowMetrix System

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Abstract
Our knowledge of the multiplicity and functions of cytokines has expanded greatly during the last 10 years. However, the large number and low concentrations of cytokines that are expressed in a pathogenic condition often preclude determining the presence and function of each cytokine during disease pathogenesis (1)(2) . The FlowMetrixTM system has been developed as a highly sensitive, multiplexed assay platform that provides a means to determine qualitative and quantitative measurements of multiple analytes simultaneously. The system utilizes polystyrene microspheres as a vehicle for different molecular reactions to occur. Differential staining of these microspheres with orange-emitting and red-emitting fluorochromes produces an array of 64 individually addressable populations of microspheres (3) . A green-emitting fluorochrome is coupled to detection molecules to measure each molecular reaction. A graphical representation of the classification of each microsphere set and the subsequent measurement of green fluorescence associated with each set is illustrated in Fig. 1 A. The use of a standard flow cytometer and its power to analyze the concentrations of three different fluorochromes on individual particles allow the simultaneous quantification of up to 64 different cytokines or chemokines in a single tissue or fluid sample in real time. We have assembled a number of different multiplexed cytokine panels to analyze proinflammatory cytokines and immunity facilitative cytokines. These panels can quantitate multiple cytokines simultaneously in a sample volume as little as 50 μL with a detection limit of at least 10 ng/L and a dynamic range of three logs or greater. The rapid kinetics of microsphere-based assays (4) as well as the homogeneous assay format allows this analysis to be completed in 30–60 min. Additionally, the inclusion of new or additional cytokines or chemokines to this panel only requires the addition of new microsphere sets.