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Free keywords:
flow cytometry; LPS; whole blood; cell activation; EDTA; heparin; intracellular TNF-α; CD69; HLA-DR
Abstract:
Background: The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed. Methods: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-α). Results: LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-α and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-α in monocytes and CD69 in lymphocytes was more efficient in heparinized blood. Conclusion: Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood. (C) 2002 Wiley-Liss, Inc.
