The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases

ten Oever, Jaap; Kox, Matthijs; van de Veerdonk, Frank L; Mothapo, Khutso M; Slavcovici, Adriana; Jansen, Tim L; Tweehuysen, Lieke; Giamarellos-Bourboulis, Evangelos J; Schneeberger, Peter M; Wever, Peter C; Stoffels, Monique; Simon, Anna; van der Meer, Jos WM; Johnson, Melissa D; Kullberg, Bart-Jan; Pickkers, Peter; Pachot, Alexandre; Joosten, Leo AB; Netea, Mihai G

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy individuals after informed consent. Briefly, PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Zeist, The Netherlands) and collecting the white interphase. Cells were washed twice in cold PBS and concentrations were adjusted to 5 × 10 6 cells/ml in RPMI-1640, supplemented 2 mM L-glutamine, 1 mM pyruvate and 50 μg/ml gentamicin (GIBCO Invitrogen, Carlsbad, CA). Mononuclear cells (5 × 10 5 ) in a 100-μl volume were added to round-bottom 96-well plates (Greiner, Nurnberg, Germany) and incubated with either 100 μl of culture medium (negative control), or LPS from E. coli O55:B5 (10 μg/ml; Sigma-Aldrich, St Louis, MO), Pam3Cys (10 μg/ml) or heat-killed E. coli ATCC 35218 (10 7 micro-organisms/ml). After 24 hour incubation at 37°C, the supernatants were stored at -80°C until measurement of sTLR2, sTLR4 and IL-6.