title: The detection of intracytoplasmic interleukin-2 in Jurkat E6.1 and human peripheral blood mononuclear cells using direct conjugate, two-colour, immunofluorescent flow cytometry.
abstract: The most commonly used approaches for the estimation of cytokine protein production involve the quantification of cytokines produced, and accumulated, in a complex body fluid or supernatant of cultured cells, by means of a bioassay or immunoassay, but these techniques do not permit an estimation of the frequency or phenotype of cytokine-producing cells. Traditional methods use immunohistochemical based techniques which can be difficult to perform and interpret, whereas flow cytometry has the advantage of objective assessment and standardisation and is less labour intensive. In this study we have established a rapid and sensitive technique for the simultaneous detection of intracellular IL-2 in conjunction with CD3. Polyclonal goat anti-IL-2 and control goat IgG were conjugated to FITC and then separated from free fluorochrome using column chromatography. PHA activated PBMC or Jurkat E6.1 cells were fixed in paraformaldehyde and permeabilised with saponin, followed by the addition of directly conjugated antibodies (FITC anti-IL-2 or PE anti-CD3) alone or in combination. Samples were then analysed using a flow cytometer and the percentage of dual labelled cells calculated. Several methods have been previously established for the detection of intracellular cytokines using flow cytometry and employing multiple layers of antibodies in the detection steps. By using direct conjugates the technique is less time consuming, requires fewer controls and can be used to examine cytokine production by identifiable cell phenotypes in a mixed cell population.
authors.affiliation: Institute for Cancer Studies, University of Sheffield Medical School, UK.
authors.forename: C A
authors.initials: CA
authors.lastname: McIntyre
authors.forename: C J
authors.initials: CJ
authors.lastname: Horne
authors.forename: J
authors.initials: J
authors.lastname: Lawry
authors.forename: R C
authors.initials: RC
authors.lastname: Rees
mesh_terms: D017252:CD3 Complex / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D003593:Cytoplasm; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D016130:Immunophenotyping; D007376:Interleukin-2 / Q000032:analysis*; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D017252:CD3 Complex; D007074:Immunoglobulin G; D007376:Interleukin-2; D016650:Fluorescein-5-isothiocyanate