       Document 0691
 DOCN  M9640691
 TI    Detection of intracytoplasmic cytokine using flow cytometry and directly
       conjugated anti-cytokine antibodies.
 DT    9604
 AU    Prussin C; Metcalfe DD; Allergic Diseases Section, National Institute of
       Allergy and; Infectious Diseases, Bethesda, MD 20892-1888, USA.
 SO    J Immunol Methods. 1995 Dec 15;188(1):117-28. Unique Identifier :
       AIDSLINE MED/96140787
 AB    Recently, there have been several reports demonstrating improvements in
       the flow cytometric detection of intracellular cytokines. These
       advances, although significant, have not yielded techniques that have
       easily been translated into broad use. To address this issue, we have
       coupled a fixation and permeabilization method with the use of directly
       labelled monoclonal anti-cytokine antibodies, providing both improved
       signal and simpler staining. The kinetics of in situ cytokine production
       in both CD4 and CD8 cells are shown for IL-2, IL-4, IL-5 and IFN-gamma.
       Based on these data, 6 h was chosen for optimal detection of this
       combination of cytokines. We show the specificity of this technique by
       blocking cytokine staining using a molar excess of recombinant cytokine.
       Additionally, unlabelled anti-cytokine antibodies are demonstrated to
       block specific staining of labelled antibody, providing an objective
       means to place statistical markers. Using such controls, we routinely
       detected as few as 0.1% false positive cells, allowing the flow
       cytometric detection of IL-5, which is below the threshold of detection
       of published methods. To further prove the specificity of staining, we
       stained using two anti-IL-5 mAbs known to recognize different epitopes
       and demonstrate that the same cells stain with both antibodies. Without
       permeabilization we could detect a fraction of cells with low intensity
       staining for cytokine. This staining was further examined using
       differential two color staining for intracellular and extracellular
       cytokine, clearly demonstrating no cells staining exclusively for
       extracellular cytokine, confirming a lack of passive transfer of
       cytokine to nearby cells. We show that cytokine flow cytometry is useful
       in examining the increased IL-5 production characteristic of
       eosinophilic states and that IL-5 production is limited to the CD27
       negative subpopulation. These data illustrate the unique capability of
       cytokine flow cytometry to correlate cytokine expression with cell
       surface phenotype without cell separation. In summary, using directly
       conjugated anti-cytokine antibodies, cytokine flow cytometry becomes a
       specific and versatile technique for the assessment of complex cytokine
       production phenotypes in fresh ex vivo T cell subpopulations.
 DE    *Antibodies/PHARMACOLOGY  Antibodies, Monoclonal/PHARMACOLOGY  Antibody
       Specificity  Binding Sites, Antibody  Binding, Competitive/IMMUNOLOGY
       Cytokines/*ANALYSIS/*IMMUNOLOGY/PHARMACOLOGY
       Cytoplasm/*CHEMISTRY/IMMUNOLOGY  CD4 Lymphocyte Count
       Eosinophilia/IMMUNOLOGY  Flow Cytometry  Fluorescent Antibody Technique,
       Direct  Human  Interleukin-5/ANALYSIS  Kinetics  Membrane
       Proteins/ANALYSIS  Recombinant Proteins/PHARMACOLOGY  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

