Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in whole blood

Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in whole blood

Autor Brunialti, Milena Karina Coló Autor UNIFESP Google Scholar
Kallas, Esper Georges Autor UNIFESP Google Scholar
Freudenberg, M. Google Scholar
Galanos, C. Google Scholar
Salomão, Reinaldo Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Max Planck Inst Immunbiol
Resumo 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-alpha). 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-alpha 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-alpha 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.
Palavra-chave flow cytometry
LPS
whole blood
cell activation
EDTA
heparin
intracellular TNF-alpha
CD69
HLA-DR
Idioma Inglês
Data de publicação 2002-02-15
Publicado em Cytometry. New York: Wiley-liss, v. 50, n. 1, p. 14-18, 2002.
ISSN 0196-4763 (Sherpa/Romeo, fator de impacto)
Publicador Wiley-Blackwell
Extensão 14-18
Fonte http://dx.doi.org/10.1002/cyto.10049
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000173969700003
Endereço permanente http://repositorio.unifesp.br/handle/11600/26756

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