title: Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers.
abstract: Chemical agents (DTT, EDTA) and enzymes (collagenase, DNAse) are often used for the generation of a single cell suspension from tissue samples. In this study, flow cytometry was used to examine the effect of chemical agents and enzymes on the expression of cell membrane markers of T lymphocytes from tonsils and peripheral blood. Expression of CD4, CD8, CD25, CD38, L-selectin, CD44, alphaEbeta7 and alpha4beta7 were studied. Incubation of lymphocytes with DTT and EDTA resulted in a decrease of CD38, alphaEbeta7 and alpha4beta7 expression. Incubation with collagenase A and DNAse resulted in a decrease of CD25, L-selectin, alphaEbeta7 and alpha4beta7. The results of this study indicate that a careful interpretation is necessary for phenotypic descriptions of lymphocyte populations obtained by enzymatic isolation techniques.
authors.affiliation: Department of Rheumatology, Ghent University Hospital, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium. Nancy.VanDamme@rug.ac.be
authors.forename: N
authors.initials: N
authors.lastname: Van Damme
authors.forename: D
authors.initials: D
authors.lastname: Baeten
authors.forename: M
authors.initials: M
authors.lastname: De Vos
authors.forename: P
authors.initials: P
authors.lastname: Demetter
authors.forename: D
authors.initials: D
authors.lastname: Elewaut
authors.forename: H
authors.initials: H
authors.lastname: Mielants
authors.forename: G
authors.initials: G
authors.lastname: Verbruggen
authors.forename: C
authors.initials: C
authors.lastname: Cuvelier
authors.forename: E M
authors.initials: EM
authors.lastname: Veys
authors.forename: F
authors.initials: F
authors.lastname: De Keyser
mesh_terms: D000293:Adolescent; D000328:Adult; D015703:Antigens, CD / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002648:Child; D002675:Child, Preschool; D017364:Collagenases / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D016023:Integrins / Q000378:metabolism; D019041:L-Selectin / Q000378:metabolism; D008875:Middle Aged; D014066:Palatine Tonsil / Q000166:cytology* / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D007202:Indicators and Reagents; D016023:Integrins; D019041:L-Selectin; D004492:Edetic Acid; D003851:Deoxyribonucleases; D017364:Collagenases; D004229:Dithiothreitol