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M9640691.TXT
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1996-03-04
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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).