Flow cytometric determination of E-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1 in formaldehyde-fixed endothelial cell monolayers.
BACKGROUND
Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1).


METHODS
TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells.


RESULTS
HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules.


CONCLUSIONS
The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.
Gräbner
R
R
Center of Vascular Biology and Medicine, Friedrich Schiller University of Jena, Erfurt, Germany. graebner@zmkh.ef.uni-jena.de
Till
U
U
Heller
R
R
D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D019040:E-Selectin / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005557:Formaldehyde; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000187:drug effects / Q000378:metabolism*; D012503:Saponins / Q000494:pharmacology; D016707:Tissue Fixation; D014357:Trypsin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins; D019010:Vascular Cell Adhesion Molecule-1 / Q000187:drug effects / Q000378:metabolism*; D014746:Vimentin / Q000378:metabolism
D019040:E-Selectin; D012503:Saponins; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D014746:Vimentin; D018799:Intercellular Adhesion Molecule-1; D005557:Formaldehyde; D014357:Trypsin