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.
van Kessel
K P
KP
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
Park
C T
CT
Wright
S D
SD
D002448:Cell Adhesion / Q000502:physiology*; D005340:Fibrinogen / Q000378:metabolism; D005452:Fluoresceins; D005470:Fluorometry / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D016023:Integrins / Q000502:physiology*; D016177:Macrophage-1 Antigen / Q000502:physiology; D009504:Neutrophils / Q000502:physiology*; D013388:Succinimides
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D016023:Integrins; D016177:Macrophage-1 Antigen; D013388:Succinimides; D005340:Fibrinogen