LIMITED LOSS OF 9 TUMOR-ASSOCIATED SURFACE ANTIGENIC DETERMINANTS AFTER TRYPTIC CELL-DISSOCIATION

Disodium ethylenediaminetetraacetic acid (EDTA) or trypsin/EDTA are fr equently used for the dispersion of monolayer cells into single cell s uspensions allowing now cytometric analysis of surface antigenic deter minants, A disadvantage of EDTA is the slow action of this agent, wher eas trypsin might affect the antigenic determinants under focus, We st udied the possible deleterious effect of trypsin on three different ov arian carcinoma cell lines, COV413b, COV362.c14, and NIH:OVCAR-3, on c ell surface antigenic determinants by now cytometry, Either EDTA or tr ypsin/EDTA was used for detachment and dissociation of monolayer ovari an cancer cell lines, followed by indirect immunofluorescence with a p anel of monoclonal antibodies directed against nine different surface antigenic determinants, including six markers directed against widely distributed antigens, Compared to EDTA, trypsin/EDTA resulted in highe r total cell yields and rapid detachment and dissociation into single cell suspensions with significantly lower amounts of dead cells detect ed by both trypan blue and propidium iodide (PI), Large differences in antigen expression were observed for the different cell lines, Howeve r, all antigenic determinants tested could still be detected after try ptic proteolysis, Three antigenic determinants were significantly decr eased after trypsin/EDTA compared to EDTA detachment, CA 125 was decre ased on COV362.c14 and NIH:OVCAR-3 cells, respectively, BMA 180 and IC AM-1 were decreased on COV413b cells, This cell line-dependent decreas e might be caused by differences in glycosylation, We conclude that tr ypsin/EDTA can be used for rapid monolayer cell detachment with high c ell yields and limited loss of antigenic determinants tested. (C) 1995 Wiley-Liss, Inc.