Reevaluation of Trypsin-EDTA for Endothelial Cell Detachment before Flow Cytometry Analysis

Abstract

To obtain viable single-cell suspensions for flow cytometry analysis, endothelial cells are usually harvested by using a solution of trypsin-EDTA or PBS-EDTA alone. Trypsin is known to alter antigenic epitopes and thus may lead to an inaccurate assessment of cell-surface molecule expression. First it was determined that the best viable cell recovery was obtained with trypsin-EDTA compared to alternative methods, i.e., cell scraper, EDTA and a cell-dissociation solution. After cell detachment with trypsin-EDTA of increasing concentrations and incubation times and indirect immunolabeling with monoclonal antibodies, flow cytometry analysis of endothelial cell antigen expression established that a solution of 0.05% trypsin-0.02% EDTA incubated for 0.5 min yielded endothelial cells whose integrity of antigenic expression was maintained. The following cell-surface molecules were studied: S-Endo 1 antigen, CD54 ([CAM-]), Thrombomodulin (TM), CD31 (PECAM-I), CD49b (VLAa2) and CD51 (VNRa).