Mec hydrochloride

U937 cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were cultured in RPMI 1640 (Gibco by Life Technologies GmbH, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS; Biochrome, Berlin, Germany) and 2 mM L-glutamine (Gibco by Life Technologies GmbH) under 5% CO₂ atmosphere at 37 °C.
To investigate IL-1β release cells were transferred to 24-well plates (1 × 10⁶ cells/ml and per well). Cells were primed with 1 μg/ml LPS from Escherichia coli (L2654; 1 μg/ml; Sigma-Aldrich, Deisenhofen, Germany) for 5 h. After priming, the P2X7 receptor agonist BzATP (Sigma-Aldrich; 100 μM) was added for 30 min in presence or absence of different concentrations of cholinergic agonists and antagonists. Cho chloride (100 μM), PC chloride calcium salt tetrahydrate (100 μM), and Mec hydrochloride (100 μM) were purchased from Sigma-Aldrich. An analogue of α-conotoxin RgIA (RgIA4)22 was used in concentrations from 0.2 to 200 nM. After cell treatment, cells were spun down (500 g, 8 min) the supernatants were collected and stored at −20 °C. IL-1β concentrations were measured using a human Quantikine Immunoassays (R&D Systems, Minneapolis, MN) and LDH was determined.

Nicotine, MEC hydrochloride, MLA citrate and DAB substrate were purchased from Sigma-Aldrich (St. Louis, MO, USA) and DHβE hydrobromide from Tocris (Bristol, UK). The primary antibody against c-Fos was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and the secondary biotinylated anti-goat IgG antibody, ABC kit from Vector Laboratories (Burlingame, CA, USA). Others common laboratory reagents were also obtained from commercial sources.