Application of X-ray microanalysis to study of the expression of endothelial adhesion molecules on human umbilical vein endothelial cells in vitro.
A semi-quantitative procedure is described, which allows the evaluation of expression levels of endothelial adhesion molecules on cultured human umbilical vein endothelial cells (HUVEC) using energy dispersive X-ray microanalysis (EDX). As a model two adhesion molecules, E-selection (CD62E; ELAM-1/endothelial leukocyte adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1; CD54), were localized by the use of the silver-enhancement colloidal gold method after stimulation of HUVEC with endotoxin lipopolysaccharide (LPS), tumour necrosis factor (TNF) or a phorbol ester (PMA). The analysis was performed in a scanning electron microscope (SEM) at an accelerating voltage of 15 kV with scanned areas of 200 x 400 microns. The semi-quantitative data indicated that in LPS-treated groups both adhesion molecules were expressed at a significantly higher level than in all other groups (P < 0.01). In addition, after a 4 h treatment the expression levels of E-selectin in all groups were higher compared to ICAM-1. The experimental data from X-ray microanalysis were compared with data obtained from an enzyme-linked immunosorbent assay (ELISA) and similar values were found for both types of preparation. This microanalytical method is relatively simple and seems to be suitable for immunogold labelling studies on different types of endothelial cells in vitro.
Tomczok
J
J
Institute of Pathology, Johannes Gutenberg University Mainz, Germany.
Sliwa-Tomczok
W
W
Klein
C L
CL
Bittinger
F
F
Kirkpatrick
C J
CJ
D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D019040:E-Selectin; D004577:Electron Probe Microanalysis; D004730:Endothelium, Vascular / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D013755:Tetradecanoylphorbol Acetate