title: Apparent loss of 2H4+ T cells in peripheral blood lymphocytes of normal donors: a 2H4-specific artefact unique to T cells.
abstract: The antigenic cluster designated CD45R is recognized by a family of monoclonal antibodies. However, one of those most frequently used because of its commercial availability is 2H4. In a series of experiments we show that 2H4 immunofluorescence is almost completely lost from CD4+ or CD8+ T cells of human origin if they are fixed after staining with 2H4. Loss of fluorescence intensity of the total population of 2H4+ lymphocytes and nearly complete quantitative loss of CD45R+ T cells is observed after fixation. This apparent loss of CD45R as detected by 2H4 is not seen if other CD45R-specific monoclonal antibodies are used. Fixation of B cells previously stained with 2H4 gives rise to slightly diminished fluorescence intensity, but no reduction in number of 2H4+ B cells. This appears to be due to a lower antigen density on T cells as compared to B cells. The effect is unique to 2H4, as other monoclonal antibodies recognizing CD45R, while exhibiting a decrease in fluorescence intensity after fixation, still unequivocally detect CD45R+ CD4+ or CD8+ T cells. The effect is restricted to human T cells, as primate lymphocytes, fixed after staining with 2H4, show no loss in fluorescence intensity. We conclude that if it is necessary to fix human T cells after fluorescent staining, the use of CD45R-specific antibodies other than 2H4 is mandatory.
authors.affiliation: Department of Immunology, University of Alberta, Edmonton, Canada.
authors.forename: H M
authors.initials: HM
authors.lastname: Serra
authors.forename: J A
authors.initials: JA
authors.lastname: Ledbetter
authors.forename: D
authors.initials: D
authors.lastname: Neil
authors.forename: L M
authors.initials: LM
authors.lastname: Pilarski
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation / Q000302:isolation & purification; D001402:B-Lymphocytes / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D006801:Humans; D017493:Leukocyte Common Antigens; D008252:Macaca fascicularis / Q000276:immunology; D013601:T-Lymphocytes / Q000145:classification / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D005404:Fixatives; D017493:Leukocyte Common Antigens