title: Limited loss of nine tumor-associated surface antigenic determinants after tryptic cell dissociation.
abstract: Disodium ethylenediaminetetraacetic acid (EDTA) or trypsin/EDTA are frequently used for the dispersion of monolayer cells into single cell suspensions allowing flow cytometric analysis of surface antigenic determinants. A disadvantage of EDTA is the slow action of this agent, whereas trypsin might affect the antigenic determinants under focus. We studied the possible deleterious effect of trypsin on three different ovarian carcinoma cell lines, COV413b, COV362.c14, and NIH:OVCAR-3, on cell surface antigenic determinants by flow cytometry. Either EDTA or trypsin/EDTA was used for detachment and dissociation of monolayer ovarian cancer cell lines, followed by indirect immunofluorescence with a panel 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 higher total cell yields and rapid detachment and dissociation into single cell suspensions with significantly lower amounts of dead cells detected by both trypan blue and propidium iodide (PI). Large differences in antigen expression were observed for the different cell lines. However, all antigenic determinants tested could still be detected after tryptic proteolysis. Three antigenic determinants were significantly decreased after trypsin/EDTA compared to EDTA detachment. CA 125 was decreased on COV362.c14 and NIH: OVCAR-3 cells, respectively. BMA 180 and ICAM-1 were decreased on COV413b cells. This cell line-dependent decrease might be caused by differences in glycosylation. We conclude that trypsin/EDTA can be used for rapid monolayer cell detachment with high cell yields and limited loss of antigenic determinants tested.
authors.affiliation: Department of Pathology, University of Leiden, The Netherlands.
authors.forename: W E
authors.initials: WE
authors.lastname: Corver
authors.forename: C J
authors.initials: CJ
authors.lastname: Cornelisse
authors.forename: J
authors.initials: J
authors.lastname: Hermans
authors.forename: G J
authors.initials: GJ
authors.lastname: Fleuren
mesh_terms: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D015703:Antigens, CD / Q000032:analysis / Q000276:immunology; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis* / Q000276:immunology; D051928:CD47 Antigen; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D004492:Edetic Acid; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D058973:Folate Receptors, GPI-Anchored; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000276:immunology; D010051:Ovarian Neoplasms / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000032:analysis / Q000276:immunology; D014357:Trypsin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D015703:Antigens, CD; D015295:Antigens, Tumor-Associated, Carbohydrate; D051928:CD47 Antigen; C497894:CD47 protein, human; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D018799:Intercellular Adhesion Molecule-1; D004492:Edetic Acid; D014357:Trypsin