title: Simultaneous detection of cytokine and immunophenotype at the single cell level by immunoenzymatic double staining.
abstract: The goal of this study was to establish a generally applicable immunoenzymatic method for the simultaneous detection of cytokine and immunophenotype at the single cell level. Evaluating various cell preparations and staining protocols, we found that permeabilization by saponin (0.1%) is very efficient, in combination with glutaraldehyde (0.04%) as fixative. Among various staining procedures, sequential immunoperoxidase labelling of the cytokine by use of diaminobenzidine, and detection of the immunophenotype by use of 4-chloronaphthol proved most discriminative. The typical localization of the cytokine reaction product ('Golgi staining') within the cell, and the 'ring-like' staining for the immunophenotype on the cell surface, allowed precise identification of double-labelled cells. Primary monoclonal antibodies from the same species could be used without loss of sensitivity and specificity for either or both antigens. This method thus provides the opportunity to study morphology, cytokine and immunophenotype simultaneously at the single cell level with standard equipment. Its application for the analysis of tissue samples is in progress, and may allow us to incorporate the cytokine-type as a new parameter in histopathological diagnostics.
authors.affiliation: Department of Haematology/Oncology, University of Freiburg, Germany.
authors.forename: D M
authors.initials: DM
authors.lastname: Behringer
authors.forename: B
authors.initials: B
authors.lastname: Sunderer
authors.forename: U
authors.initials: U
authors.lastname: Andersson
authors.forename: V
authors.initials: V
authors.lastname: Kresin
authors.forename: R
authors.initials: R
authors.lastname: Mertelsmann
authors.forename: A
authors.initials: A
authors.lastname: Lindemann
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D015703:Antigens, CD / Q000032:analysis; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis*; D016591:Histocytological Preparation Techniques; D006801:Humans; D007124:Immunoenzyme Techniques; D016130:Immunophenotyping / Q000379:methods*; D051379:Mice; D009000:Monocytes / Q000737:chemistry* / Q000166:cytology; D051381:Rats; D012016:Reference Values; D013601:T-Lymphocytes / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D016207:Cytokines