Title
Use of fluorescent dyes in the determination of adherence of human leukocytes to endothelial cells and the effect of fluorochromes on cellular function Use of fluorescent dyes in the determination of adherence of human leukocytes to endothelial cells and the effect of fluorochromes on cellular function
Author
Faculty/Department
Faculty of Medicine and Health Sciences
Publication type
article
Publication
Amsterdam ,
Subject
Chemistry
Biology
Human medicine
Source (journal)
Journal of immunological methods. - Amsterdam
Volume/pages
172(1994) :1 , p. 115-124
ISSN
0022-1759
ISI
A1994NQ80300012
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
Abstract
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.
E-info
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