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 dete rmine conditions of fixation and dissociation of human umbilical vein endot helial cell (HUVEC) monolayers that permit a reliable now cytometric determ ination of intracellular and surface content of E-selectin, vascular cell a dhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) . Methods: TNF alpha-treated HUVEC monolayers were fixed with 0.5% formaldehy de at the end of the experimental incubation. Subsequently, either the mono layer was trypsinized and thereafter the cells were subjected to indirect f luorescence labeling or the monolayer was first labeled and then dissociate d by trypsinization. Cell integrity was assessed by vimentin staining. Tota l adhesion molecule content was detected in saponin-permeabilized cells. Results: HUVEC integrity was maintained when the fixation time of the monol ayer did not exceed 5 min and trypsin/EDTA was used fur dissociation. Surfa ce adhesion molecules were partially hydrolyzed by trypsin when trypsinizat ion preceded labeling but antibody binding protected adhesion molecules fro m degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitiv e surface fractions but surface ICAM-1 was mainly trypsin resistant. Permea bilization with 0.06 saponin allowed the detection of considerable intracel lular pools of the investigated adhesion molecules. Conclusions: The described method permits the reliable determination of sur face and intracellular fractions of adhesion molecules in formaldehyde-fixe d HUVEC monolayers and may be used for studies on the regulation of adhesio n molecule expression. (C) 2000 Wiley Liss, Inc.