van Kessel K P, Park C T, Wright S D
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
J Immunol Methods. 1994 Jun 3;172(1):25-31. doi: 10.1016/0022-1759(94)90375-1.
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.
中性粒细胞（PMN）上白细胞整合素CR3（Mac-1，CD11b/CD18，αmβ2）的亲和力可被多种激动剂快速调节。我们描述了一种通过测量PMN与纤维蛋白原包被表面的黏附来确定这些受体亲和力的方法。用羧基荧光素二乙酸琥珀酰亚胺酯加载细胞，其被细胞内酯酶脱乙酰化，产生高度荧光的羧基荧光素，该荧光素被困在PMN内。用荧光酶标仪对黏附在Terasaki微孔板纤维蛋白原包被孔中的细胞数量进行定量。用多种激动剂刺激PMN，包括佛波酯、甲酰甲硫氨酰-亮氨酰-苯丙氨酸、钙离子载体A23187、白细胞介素-8、肿瘤坏死因子和脂多糖，可强烈增加对纤维蛋白原的黏附，这依赖于CD11b/CD18。细胞黏附程度取决于刺激浓度和孵育时间。该测定所需时间少，使用细胞数量少，且不需要危险试剂。
