Lta sa

LTA-SA, FSL-1, FLA-ST, poly(I:C), LPS, R837, R848, CpG-A (ODN 2216) and CpG-B (ODN 2006) were purchased from Invivogen.

Blood samples were collected in tubes containing heparin and were tested, generally after 24 hours of transport at room temperature. Blood was diluted 1:2 in RPMI 1640 medium (Gibco BRL, Invitrogen, USA). Aliquots of diluted blood were dispensed into a 48-well plate and incubated at 37°C, under an atmosphere containing 5% CO₂, under different sets of conditions: with medium alone (non-stimulated), with agonists of different receptors (20 ng/ml IL-1β (R&D Systems), 10 ng/ml TNF-α (R&D Systems), 25 μg/ml poly(I:C) (polyinosinic-polycytidylic acid from GE Healthcare) and TLR2 ligands: 1 ug/ml PAM₂CSK₄, 1 ug/ml PAM₃CSK₄, 1 μg/ml purified LTA-SA, 100 ng/ml FSL-1, 500 ug/ml of lipidA (all from Invivogen), 1 ng/ml of LPS Salmonella enterica serotype minnesota Re 595 (Sigma) or with 10^-7 M PMA/ 10^-5 M ionomycin (as positive control of stimulation) in presence of 10ug/ml of polymixin B. Supernatants were collected after 48 hours and stored at -20°C until their use for cytokine determinations by ELISA. Exogenous monoclonal anti-LTA Ab (Pagibaximab) was obtained through collaboration with Biosynexus Incorporated (Gaithersburg, MD, USA) and palivizumab (Synagis®, Abbott) was used as an isotypic control. These mAb were used at 0.5 or 1mg/ml.