Pc chloride calcium salt tetrahydrate

U937 cells (DSMZ, Braunschweig, Germany) were maintained in RPMI 1640 (Gibco by Life Technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS, Biochrome, Berlin, Germany) and 2 mM l-glutamine (Gibco by Life Technologies) under 5% CO₂ atmosphere at 37°C. Cells (1 × 10⁶ cells/ml) were seeded in 24-well plates, primed for 5 h with 1 µg/ml lipopolysaccharide (LPS) from Escherichia coli (L2654, Sigma-Aldrich, Deisenhofen, Germany) (30 (link)). BzATP [2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt; 100 µM, Sigma-Aldrich] or nigericin (50 µM, Sigma-Aldrich) were added for another 30 min in the presence or absence of different concentrations of eCRP from human pleural fluid (Millipore, AG732), recombinant CRP (rCRP) produced in E. coli (Millipore, 236608), serum amyloid P (SAP; Millipore, 565190), or PC chloride calcium salt tetrahydrate (Sigma-Aldrich). Nicotinic antagonists mecamylamine hydrochloride (Sigma-Aldrich), strychnine hydrochloride (Sigma-Aldrich), α-bungarotoxin (Tocris Bioscience, Bristol, UK), ArIB [V11L, V16D] (500 nM) (34 (link), 35 (link)) and RgIA4 (200 nM) (31 (link), 36 (link)) were also applied together with BzATP. Supernatants were stored at 20°C until cytokine and lactate dehydrogenase (LDH) measurement.

Mouse peripheral blood mononuclear cells (PBMCs) were freshly isolated from heparinized blood obtained from WT, Chrna7, Chrna9, or Chrna10 gene-deficient mice using Percoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden; 1.082 g/ml) density gradient centrifugation. Mononuclear cells were cultured for 2 h in 24-well-plates at 37°C, 5% CO₂, in RPMI 1640 (Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS Superior EU, Biochrom GmbH, Berlin, Germany) and 2 mM L-glutamine (GlutaMAX™, Gibco/Life Technologies). Thereafter, cells were stimulated with 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate trieethylammonium salt (BzATP, 100 μM, Sigma-Aldrich, Taufkirchen, Germany) for 30 min, in the presence or absence of ACh chloride (10 μM), PC chloride calcium salt tetrahydrate (PC, 100 μM) or nicotine (Nic, 100 μM; all purchased from Sigma-Aldrich, Taufkirchen, Germany). Cell culture supernatants were collected and stored at −20°C until IL-1β and lactate dehydrogenase (LDH) measurement.

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.