Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers.
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.
Van Damme
N
N
Department of Rheumatology, Ghent University Hospital, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium. Nancy.VanDamme@rug.ac.be
Baeten
D
D
De Vos
M
M
Demetter
P
P
Elewaut
D
D
Mielants
H
H
Verbruggen
G
G
Cuvelier
C
C
Veys
E M
EM
De Keyser
F
F
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*
D015703:Antigens, CD; D007202:Indicators and Reagents; D016023:Integrins; D019041:L-Selectin; D004492:Edetic Acid; D003851:Deoxyribonucleases; D017364:Collagenases; D004229:Dithiothreitol