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Influence of EDTA and Heparin on Lipopolysaccharide Binding and Cell Activation, Evaluated at Single-Cell Level in Whole Blood

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons191059

Freudenberg,  Marina
Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons191064

Galanos,  Chris
Emeritus Group: Cellular Immunology, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Citation

Brunialti, M. K. C., Kallás, E. G., Freudenberg, M., Galanos, C., & Salomao, R. (2002). Influence of EDTA and Heparin on Lipopolysaccharide Binding and Cell Activation, Evaluated at Single-Cell Level in Whole Blood. Cytometry, 50(1), 14-18.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002B-9667-D
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.