Tlrgrade

Neutrophils were isolated from the BM and cultured in for 24 h in presence of 2 μg/mL LPS (from E. coli, Serotype R515 (Re), TLR grade, ENZO Life Sciences) or 1 x 10⁶ particles/mL S. aureus bioparticles. Supernatants were added to freshly isolated LN B cells. Isolated iLN B cells were cultured in complete lymphocyte medium at initial concentration between 5 x 10⁵ to 2 x 10⁶ cells/mL. B cells and neutrophils were added to the cultures in ratio 10 B cells: 1 neutrophil, for 5 days in presence of LPS or S. aureus bioparticles. Alternatively, supernatants from either activated or non-activated neutrophil cultures (25% of culture medium) were added to B cells. Final concentration of LPS was 2 μg/mL, and S. aureus 1 x 10⁶ particles/mL in all cultures. Neutralizing anti TGF-β1 (TGF-beta 1/1.2 Polyclonal antibody; R&D Systems) were added at final concentration 1 μg/mL, and TGF-β1 (50 ng/mL). TGF-β1 in neutrophil and B cell cultures was measure using commercial ELISA kit (eBioscience). IgM and IgA levels in the supernatants were measured with commercial ELISA kits (eBioscience) according to the manufacturer's protocol.

Primary blood monocytes were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat inactivated, low endotoxin fetal calf serum (HI FCS-LE, Gibco), 1% (v/v) Penicillin-Streptomycin (P/S) and 1% (v/v) L-glutamine. Subsets of monocytes at 12,500 cells per well (50,000 cells/ml) were plated in 96 well plates and stimulated with 1 ng/ml LPS (purified from E. coli, serotype 0111:B4, TLRgrade, Enzo Life Sciences) for 24 h either in the presence or absence of 10 μM A438079 hydrochloride (P7X7R antagonist, Tocris Bioscience). 300 μM BzATP (P2X7R agonist, Sigma-Aldrich) was added for the final 20 minutes of incubation. Culture supernatants were collected and non-adherent cells removed by centrifugation. Samples were stored at −80 °C until analysis.