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New tools that facilitate the study of cell-to-cell variability could help uncover novel cellular regulation mechanisms. We present an integrated microfluidic platform to analyze a large number of single cells in parallel. To isolate and analyze thousands of individual cells in multiplexed conditions, our platform incorporates arrays of microwells (7 pL each) in a multilayered microfluidic device. The device allows the simultaneous loading of cells into 16 separate chambers, each containing 4640 microwells, for a total of 74 240 wells per device. We characterized different parameters important for the operation of the microfluidic device including flow rate, solution exchange rate in a microchamber, shear stress, and time to fill up a single microwell with molecules of different molecular weight. In general, after ~7.5 min of cell loading our device has an 80% microwell occupancy with 1-4 cells, of which 36% of wells contained a single cell. To test the functionality of our device, we carried out a cell viability assay with adherent and nonadherent cells. We also studied the production of neutrophil extracellular traps (NETs) from single neutrophils isolated from peripheral blood, observing the existence of temporal heterogeneity in NETs production, perhaps having implications in the type of the neutrophil response to an infection or inflammation. We foresee our platform will have a variety of applications in drug discovery and cellular biology by facilitating the characterization of phenotypic differences in a monoclonal cell population. [ABSTRACT FROM AUTHOR]