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Published on: September 15, 2017
Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in
Milena Karina Coló Brunialti1, Esper Georges Kallás, Marina Freudenberg
1Division of Infectious Diseases, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
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.
