Lps salmonella enterica

Peritoneal macrophages were obtained from the peritoneal cavity of 8-week-old male NMRI mice 4 h after i.p. endotoxin (lipopolysaccharide: LPS) injection (300 µL of 300 µg/mL solution per animal, Salmonella enterica LPS, Sigma-Aldrich, St. Louis, MO, USA). Four hours later mice were exsanguinated under deep ketamine and xylazine anaesthesia. The abdominal cavity was leached using 3 mL cell culture medium (RPMI 1640, Sigma, St. Louis, MO, USA) supplemented with 10% foetal bovine serum under sterile conditions. The lavage fluid was collected into ice-cold tubes and cell count was determined in the samples with a haemocytometer. In the next step 100 μL lavage fluid samples, 1 µL LPS solution and 100 µL of test compounds were added into 800 μL culture medium in a 24-well plate. The plates were then incubated in CO₂-incubator at 37 °C. Well contents were collected 8 h later and centrifuged for 5 min at 12,500 rpm. The concentration of the inflammatory cytokine IL-1β was measured from the supernatants by sandwich ELISA method using IL-1β BD OptEIA ELISA set (cat. Nr. 559603, BD Biosciences Eastern Europe, Heidelberg, Germany) [46 (link)].

Murine RAW 264.7-Blue cells (Invivogen) derived from the murine RAW 264.7 macrophages and stably expressing an NF-κB-inducible secreted alkaline phosphatase (SEAP) reporter gene were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% Fetal bovine serum (FBS), L-Glutamine (2 mM) and sodium pyruvate (0.4 mM), in the presence of selection antibiotic Zeocin (200 μg/mL). E. coli O111:B4 LPS was purchased from Invivogen. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. Compounds FP13-17 were reconstituted in DMSO/ethanol to provide a 10 mM stock solution. Further dilutions were made in cell culture medium so that the final amount of DMSO in the cell culture did not exceed 0.05%. C57BL6/J mice were obtained from Janvier. All animal experiments were approved by the animal ethical committee of Ghent University, Faculty of Sciences. All methods were performed in accordance with the relevant guidelines and regulations. Salmonella enterica LPS used for in vivo studies was purchased from Sigma-Aldrich.