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Computerized assessment of production of multiple human cytokines at the single‐cell level using image analysis
Author(s) -
Björk Lars,
Fehniger Thomas E.,
Andersson Ulf,
Andersson Jan
Publication year - 1996
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.59.2.287
Subject(s) - cytokine , biology , peripheral blood mononuclear cell , immunostaining , cell culture , tumor necrosis factor alpha , immunology , microbiology and biotechnology , cell , pathology , in vitro , immunohistochemistry , biochemistry , medicine , genetics
A technique using a computerized image analysis system was developed for evaluating and quantifying human cytokine production. This system registered single cells as positive or negative cytokine producers based on a specific juxtanuclear staining pattern generated by accumulation of the proteins in the Golgi‐endoplasmatic reticulum compartment. The characteristic morphology of the immunocytochemical staining offered the opportunity to register individual producer cells within multicomponent cell populations. A color camera was then adapted to transfer on‐line images directly into the computer‐controlled operating system. In this study cultured human peripheral blood mononuclear cells were polyclonally stimulated and then analyzed for interleukin‐1α (IL‐1α), IL‐1β, IL‐1ra, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, tumor necrosis factor‐α (TNF‐α), TNF‐β, interferon‐γ, granulocyte colony‐stimulating factor and granulocyte‐macrophage colony‐stimulating factor production. The image‐analyzing system detected cytokine‐producing cells in a sensitive and reproducible manner, which was in total congruence with enumeration by conventional microscopy. Furthermore, accurate assessments of cell distributions by signal intensity and cell area were applied at the single‐cell level. The image‐analyzing system allowed the detection of at least 1 in 1,000 events by using unique cytokine‐associated morphometric criteria. The results of kinetic studies measuring cytokine production following activation and cell transformation provided data supporting increases in intensity of intracellular localized specific immunostaining and in cell size within the cytokine‐producing cells.