Lps from escherichia coli l2654

The human histocytic lymphoma cell line U937 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and cultured according to the protocol described for mouse PBMCs. Cells in the log-phase of growth were transferred to 24-well plates (1 × 10⁶ cells/ml and per well) and primed with 1 μg/ml LPS from Escherichia coli (L2654; Sigma-Aldrich) for 5 h. Thereafter, BzATP (100 μM) was applied for 30 min, in the presence or absence of ACh (10 μM), Nic (100 μM), PC (100 μM), L-α-G-PC (Sigma-Aldrich; 0.1 μM to 1000 μM) or 1-palmitoyl-sn-glycero-3-phosphocholine LPC (C16:0), Sigma-Aldrich; 0.1 μM to 100 μM). To test the involvement of different subunits of nAChR, the following antagonists were applied: mecamylamine hydrochloride (Mec, 100 μM, Sigma-Aldrich), α-bungarotoxin (α-Bun, 1 μM, Tocris Bioscience, Bristol, UK), strychnine hydrochloride (Stry, 10 μM, Sigma-Aldrich) or conotoxins ArIB [V11L, V16D] (500 nM) or RgIA4 (200 nM; Ramarao and Cohen, 1998 (link); Whiteaker et al., 2007 (link); Innocent et al., 2008 (link); Richter et al., 2016 (link); Romero et al., 2017 (link)).

LPS from Escherichia coli (L2654) was obtained from Sigma Aldrich (Saint Louis, Missouri USA) and ultrapure LPS from Porphyromonas gingivalis from Invivo Gen (San Diego, USA); CWPS (Pneumococcal-cell-wall-polysaccharide mixture) was obtained from Statens Serum Institute (Copenhagen, Denmark), and CEF peptide pool from R&D Systems (Minneapolis, USA). RPMI 1640 with L-Glutamine, HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) and low-endotoxin fetal calf serum (FCS) were purchased from PAA (Pasching, Austria). Pairs of antibodies against IL-1β and IL-8 suitable for enzyme-linked immunosorbent spot (ELISpot) assay were obtained from BioLegend/BIOZOL (Eching, Germany). Streptavidin alkaline phosphatase (Streptavidin-ALP) was from Mabtech (Hamburg, Germany) and BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) substrate was from Sigma Aldrich (Steinheim, Germany).