Krug N, Thurau A M, Lackie P, Baier J, Schultze-Werninghaus G, Rieger C H, Schauer U
Department of Paediatrics, Ruhr University, Bochum, Germany.
J Immunol Methods. 1996 Apr 19;190(2):245-54. doi: 10.1016/0022-1759(95)00272-3.
Eosinophils and their basic proteins play a major role in allergic disease and methods are required to monitor their expression in clinical situations. In this article we describe a flow cytometric method for the detection of intracellular eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in unseparated clinical samples. After fixation with parabenzoquinone and permeabilization with n-octyl-beta-D-glucopyranoside, the detection of intracellularly stored proteins was achieved using of monoclonal antibodies against ECP (EG1, EG2) and EPO in combination with an FITC-labeled second step antibody. Confocal microscopy was used to demonstrate the intracellular origin of the fluorescent signal. Fixation with parabenzoquinone was superior to a previously described protocol using paraformaldehyde, since it reduces non-specific binding of FITC to the basic proteins in eosinophils. Fixation and permeabilization do not alter the light scatter characteristics of eosinophils in contrast to other leukocytes and thus permit gating on eosinophils without prior purification. Furthermore, the procedure does not alter the detection of cell surface antigens on eosinophils and simultaneous measurements of surface antigens and intracellular proteins is possible. We have used different clinical samples (peripheral blood, bone marrow cells) to demonstrate differences in the expression of ECP and EPO. We conclude that the detection of intracellular eosinophil proteins by flow cytometry is a rapid, easy and semiquantitative procedure which may be used to study their expression in diseases where eosinophils are involved.
嗜酸性粒细胞及其碱性蛋白在过敏性疾病中起主要作用，因此需要一些方法来监测它们在临床情况下的表达。在本文中，我们描述了一种流式细胞术方法，用于检测未分离的临床样本中的细胞内嗜酸性粒细胞阳离子蛋白（ECP）和嗜酸性粒细胞过氧化物酶（EPO）。在用对苯醌固定并用正辛基-β-D-吡喃葡萄糖苷通透后，使用针对ECP（EG1、EG2）和EPO的单克隆抗体结合异硫氰酸荧光素（FITC）标记的二抗来检测细胞内储存的蛋白质。共聚焦显微镜用于证明荧光信号的细胞内来源。与使用多聚甲醛的先前描述的方案相比，用对苯醌固定具有优势，因为它减少了FITC与嗜酸性粒细胞中碱性蛋白的非特异性结合。与其他白细胞不同，固定和通透不会改变嗜酸性粒细胞的光散射特性，因此无需事先纯化即可对嗜酸性粒细胞进行门控。此外，该方法不会改变嗜酸性粒细胞表面抗原的检测，并且可以同时测量表面抗原和细胞内蛋白质。我们使用了不同的临床样本（外周血、骨髓细胞）来证明ECP和EPO表达的差异。我们得出结论，通过流式细胞术检测细胞内嗜酸性粒细胞蛋白是一种快速、简便且半定量的方法，可用于研究它们在涉及嗜酸性粒细胞的疾病中的表达。
