One 5 ml tube was immediately centrifuged selleck catalog and plasma samples were stored at -80��C until assayed (within two months). Another 5 ml tube was used for the analysis of leukocyte-bound LPS.Isolation of peripheral blood mononuclear cells from whole bloodFor measurement of endotoxin associated with circulating leukocytes, peripheral blood mononuclear cells (PBMC) were isolated from whole blood after dilution 1:1 with RPMI-1640 (Lonza, Verviers, Belgium) and centrifugation (680 g, 15��C for 20 minutes) on Ficoll-Hypaque (Eurobio, Les Ulis, France). After centrifugation, the cells at the medium/Ficoll interface were collected, washed with RPMI-1640 and centrifuged (350 g, 10��C for five minutes). The pellet was resuspended with sterile endotoxin-free saline (0.9% sodium chloride) (Fresenius, S��vres, France).
PBMC were lysed by five cycles of freezing and thawing, and stored at -80��C until assayed (within two months).Detection of circulating NOD2 agonist in human plasmaHuman embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (PAA, Pasching, Austria). HEK293T cells were seeded into 24-well plates at a density of 105 cells/ml (500 ��l/well). The detection of NOD2 agonist in biologic fluids using this transfected cell line has been previously described . Briefly, HEK293T cells were transfected with a plasmid permitting the constitutive expression of NOD2 (sensor of both Gram-positive and Gram-negative PGN) and an NF-��B-dependent reporter gene coding for luciferase.
To the transfected cells, 50 ��l of plasma was added and then incubated for six hours. The plasma were either from patients or from healthy volunteers (ICAReB, Institut Dacomitinib Pasteur, Paris, France). The presence of NOD2 agonist in plasma was assessed by luciferase activity in cell lysates. To the cells, 100 ��l of lysis buffer (25 mM Tris-phosphate pH 8, 8 mM MgCl2, 1 mM dithiothreitol, 15% glycerol and 1% Triton X-100) was added and luciferase activity in 10 ��l of cell extracts was measured in a microplate luminometer (LB960 luminometer centro, Berthold Technologies, Germany) after the addition of 100 ��l of substrate buffer to a final concentration of 1.8 mM luciferin and 1 mM ATP. Luciferase activity was expressed as relative light unit. In some experiments, a frameshift mutant of NOD2 (fsNOD2) devoid of NF-��B activation capacity in response to NOD2 ligand was used as a negative control (kind gift of Dana J Philpott, University of Toronto, Toronto, Canada).