A novel sensitive assay to define immune status using short-term peripheral blood derived cell culture and dual-color flow cytometry.
In this study we describe a novel and highly sensitive in vitro system to determine the functionality of immune cells based on short term culture of peripheral blood derived mononuclear cells (PBMCs) and subsequent analysis of cellular proliferation and surface marker expression by automated dual-color flow cytometry. The standardized mild stimuli introduced into the culture system by supplemented medium (containing exogenous interleukin-2 (IL-2), and fetal bovine serum (FBS)) allow a more physiological interaction of the different cell subsets contained in PBMCs (including CD14+ accessory cells) than other methods that are based on potent and harsh cell activators, such as phytohemagglutinin (PHA) or anti-CD3 antibodies. Measurement of T-cell proliferation and cell surface marker (CD3, C25, CD26, CD71, HLA-DR) analysis revealed that activation response capacity in our assay depends on both the status of the obtained cells and their ability to interact in culture with CD14+ cells. This in vitro assay proved to be very sensitive in detecting changes in the status of T-cell activation and proliferation capacity, and avoid the use of radioactive reagents.
Institute of Human Virology, Medical Biotechnology Center, University of Maryland, Baltimore 21201, USA. zella@umbi.umd.edu
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Zella
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Riva
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Weichold
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Reitz
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Gerna
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD / Q000096:biosynthesis; D015415:Biomarkers; D017252:CD3 Complex / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology*; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008213:Lymphocyte Activation; D008934:Mitogens / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015415:Biomarkers; D017252:CD3 Complex; D007376:Interleukin-2; D018950:Lipopolysaccharide Receptors; D008934:Mitogens; D010835:Phytohemagglutinins; D013936:Thymidine