Davenpeck K L, Chrest F J, Sterbinsky S A, Bickel C A, Bochner B S
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
J Immunol Methods. 1995 Dec 15;188(1):79-89. doi: 10.1016/0022-1759(95)00206-5.
Fluorescently labeled leukocytes are commonly used in in vitro and in vivo experimental systems. However, the effects of fluorescent labeling on the expression and function of leukocyte adhesion molecules has not been examined in part because the extreme intensity of fluorescence tends to obscure signals from other fluorochromes used for dual color analysis. We have utilized a novel technique involving a 7-amino-4-methylcoumarin-3-acetic acid (AMCA) fluorophore-conjugated F(ab')2 fragment excitable in the ultraviolet wavelength range (350-450 nm) and dual-laser flow cytometry to determine if labeling of human neutrophils and eosinophils with the fluorescent dye 5-(6)-carboxyfluorescein diacetate (CFDA) alters surface expression of the primary leukocyte adhesion molecules involved in leukocyte-endothelial interactions. Simultaneously, adhesion molecule function was assessed by comparing the ability of CFDA-labeled vs. control cells to adhere to cultured human umbilical vein endothelial cells (HUVEC) and purified immobilized adhesion molecules. Isolated human eosinophils and neutrophils were fluorescently labeled by incubation with CFDA. Flow cytometric comparisons of labeled and unlabeled cells demonstrated that fluorescence labeling of neutrophils and eosinophils with CFDA did not alter basal surface expression of the beta 2 integrins (i.e., CD11a CD11b or, CD18). Stimulation of neutrophils with fMLP and eosinophils with PMA resulted in increased surface expression of CD11b and CD18 which was not altered by CFDA labeling. Likewise, CFDA labeling of neutrophils and eosinophils did not significantly alter their integrin-dependent adhesion to activated HUVEC under static or rotational conditions. Similarly, adhesion to immobilized recombinant E- and P-selectin was unaltered. These data demonstrate that fluorescent labeling of human neutrophils and eosinophils with CFDA does not alter surface expression or function of several adhesion molecules necessary for leukocyte-endothelial interactions. The use of CFDA-labeled cells in experiments employing intravital microscopy should therefore provide valid information on adhesion molecule function in vivo.
荧光标记的白细胞常用于体外和体内实验系统。然而，荧光标记对白细胞黏附分子表达和功能的影响尚未得到研究，部分原因是荧光强度过高往往会掩盖用于双色分析的其他荧光染料发出的信号。我们采用了一种新技术，该技术涉及一种在紫外波长范围（350 - 450 nm）可激发的7 - 氨基 - 4 - 甲基香豆素 - 3 - 乙酸（AMCA）荧光团偶联的F(ab')2片段以及双激光流式细胞术，以确定用荧光染料5 - (6) - 羧基荧光素二乙酸酯（CFDA）标记人中性粒细胞和嗜酸性粒细胞是否会改变参与白细胞 - 内皮细胞相互作用的主要白细胞黏附分子的表面表达。同时，通过比较CFDA标记的细胞与对照细胞黏附于培养的人脐静脉内皮细胞（HUVEC）和纯化的固定化黏附分子的能力来评估黏附分子的功能。分离的人嗜酸性粒细胞和中性粒细胞通过与CFDA孵育进行荧光标记。标记细胞与未标记细胞的流式细胞术比较表明，用CFDA对中性粒细胞和嗜酸性粒细胞进行荧光标记不会改变β2整合素（即CD11a、CD11b或CD18）的基础表面表达。用fMLP刺激中性粒细胞以及用PMA刺激嗜酸性粒细胞会导致CD11b和CD18的表面表达增加，而CFDA标记不会改变这种增加。同样，CFDA对中性粒细胞和嗜酸性粒细胞的标记在静态或旋转条件下不会显著改变它们对活化的HUVEC的整合素依赖性黏附。类似地，对固定化重组E - 选择素和P - 选择素的黏附也未改变。这些数据表明，用CFDA对人中性粒细胞和嗜酸性粒细胞进行荧光标记不会改变白细胞 - 内皮细胞相互作用所需的几种黏附分子的表面表达或功能。因此，在采用活体显微镜检查的实验中使用CFDA标记的细胞应该能够提供关于体内黏附分子功能的有效信息。
