Quantitative measurements of the strength of adhesion of human neutrophils to a substratum in a microfluidic device

We describe a quantitative assay of the strength of adhesion of activated and nonactivated human neutrophils to a substratum, which is carried out in a custom-made microfluidic device. The strength of adhesion is quantified by the fraction of cells remaining adherent (ACF) after a given time of exposure to shear stress in a test microchannel. The microfluidic device is made of two layers of poly(dimethylsiloxane) with integrated membrane valves. This construction allows concurrent testing of two different populations of cells, as well as setting well-defined times of exposure of cells to stress and of their incubation prior to the exposure. The test microchannels have a tapered profile, exposing cells to nearly an order of magnitude range of shear stress. ACF is measured periodically by computer-controlled videomicroscopy scans of the device, with up to 60 000 individual cells identified within a 90 seconds scan. The high throughput of the scans allows reliable quantitative assessment of the ACF. Adhesion of untreated neutrophils and neutrophils activated with formyl-Met-Leu-Phe was tested concurrently in a series of experiments with a fibrinogen-coated glass substratum. At optimized testing conditions, the ACF of activated cells was consistently found to be three times higher than that of nonactivated cells. An adhesion assay could be completed within 11 min from the loading of cells into the device without any intervention by the operator. The proposed device and assay could be used to assess the state of activation of neutrophils in human blood with a potential application to diagnostics of inflammation.