De Clerck L S, Bridts C H, Mertens A M, Moens M M, Stevens W J
Department of Immunology, Allergy and Rheumatology, University of Antwerp, Belgium.
J Immunol Methods. 1994 Jun 3;172(1):115-24. doi: 10.1016/0022-1759(94)90384-0.
A number of supravital fluorochromes are available to study leucocyte functions in vitro and in vivo. The fluorescein ester most widely used, fluorescein diacetate, has the disadvantage of rapid cellular efflux, whereas more recently developed fluorescent probes do not exhibit this inconvenient trait. However, their effect on cellular functions has not been thoroughly investigated in humans. In this study, we describe a simple and rapid fluorometric method for measuring cell adhesion to endothelium, comparing 5 different fluorochromes. Furthermore, we evaluated the effect of fluorescent dye labelling (with CFDA, CFSE, BCECF-AM, calcein-AM or DiI), on various cell functions, including, apart from adhesion, lymphocyte proliferation, granulocyte chemotaxis and superoxide production. calcein-AM and DiI proved to be the fluorochromes with the least effect on cellular function. BCECF-AM did not interfere with lymphocyte proliferation, but exhibited some influence on superoxide production and chemotaxis of granulocytes. CFDA showed a detrimental effect on both lymphocyte and granulocyte functions whereas CFSE gave intermediate results. In the adhesion assay, calcein-AM, CFSE and DiI performed comparably well. Since labelling with C12-DiI was homogeneous, this probe was also appropriate for the adhesion test, although somewhat higher background staining was present. We conclude that the fluorochromes are powerful tools when analysing the adhesion of human leucocytes to endothelial cells. However, since fluorochrome labelling can interfere with other cellular functions, the fluorescent probe has to be carefully chosen with regard to the cell type and function to be studied.
有多种超活荧光染料可用于体外和体内研究白细胞功能。最广泛使用的荧光素酯，即荧光素二乙酸酯，具有细胞快速外排的缺点，而最近开发的荧光探针则没有这种不便的特性。然而，它们对细胞功能的影响尚未在人体中进行彻底研究。在本研究中，我们描述了一种简单快速的荧光测定法，用于测量细胞与内皮细胞的粘附，比较了5种不同的荧光染料。此外，我们评估了荧光染料标记（使用CFDA、CFSE、BCECF-AM、钙黄绿素-AM或DiI）对各种细胞功能的影响，除了粘附外，还包括淋巴细胞增殖、粒细胞趋化性和超氧化物生成。钙黄绿素-AM和DiI被证明是对细胞功能影响最小的荧光染料。BCECF-AM不干扰淋巴细胞增殖，但对粒细胞的超氧化物生成和趋化性有一定影响。CFDA对淋巴细胞和粒细胞功能均有不利影响，而CFSE则表现出中等结果。在粘附试验中，钙黄绿素-AM、CFSE和DiI表现相当。由于用C12-DiI标记是均匀的，该探针也适用于粘附试验，尽管背景染色略高。我们得出结论，荧光染料在分析人类白细胞与内皮细胞的粘附中是强大的工具。然而，由于荧光染料标记可能会干扰其他细胞功能，因此必须根据要研究的细胞类型和功能仔细选择荧光探针。
