A fluorescence microassay for the quantitation of integrin-mediated adhesion of neutrophil

The avidity of leukocyte integrin CR3 (Mac-1, CD11b/CD18, alpha m beta 2) on neutrophils (PMN) may be rapidly modulated by several agonists. We describe a method for determining the avidity of these receptors by measuring the adhesion of PMN to fibrinogen-coated surfaces. Cells are loaded with a succinimidyl ester of carboxyfluorescein diacetate, which is deacetylated by intracellular esterases yielding a highly fluorescent carboxyfluorescein that remains trapped within the PMN. The number of cells adhering to fibrinogen-coated wells of Terasaki microplates is quantitated with a fluorescence plate reader. Stimulation of PMN with a number of agonists, including PMA, fNLLP, Ca-ionophore A23187, interleukin-8, tumor necrosis factor and lipopolysaccharide strongly increased adhesion to fibrinogen, which was CD11b/CD18 dependent. The extent of cell adhesion depended on stimulus concentration and incubation time. This assay requires little time, utilizes small numbers of cells and does not require hazardous reagents.