Lipopolysaccharides lps from e coli

Cells were isolated from TT by mechanical disaggregation, as described above. 0.5 × 10⁶ cells per well were seeded onto a 24-well tissue culture-treated plate and cultured in complete RPMI-1640 medium (Life Technologies). Cells were untreated or pre-treated with 100 nM of HDAC inhibitor Vorinostat (SAHA, MK0683, Selleckchem, Texas, USA) or 2 μg/ml of anti-IL-6 monoclonal antibody (Sino Biological, Beijing, China) [55 (link)]. After a 2-h incubation period, 100 ng/ml of Lipopolysaccharides (LPS from E. coli, Sigma-Aldrich) was added to all the wells. Cells were kept in a humidified incubator at 37 °C in 5% CO₂ for 5 days. Every other day, HDAC inhibitor and anti-IL-6 mAb were added to the wells at the same concentration. Cells were harvested on day 5 post treatment for RNA extraction.

RAW 264.7 cells were purchased from the American Type Culture Collection (ATCC TIB-71). Live bacterial cultures of Corynebacterium xerosis, Enterococcus faecalis, Bacillus subtilis, Staphylococcus epidermidis, Bacillus megaterium, Staphylococcus aureus, Escherichia coli and Salmonella typhimurium were purchased from Ward’s Science (Rochester, NY, USA). An additional E. coli ΔtolC mutant was also used is this study; the mutation to the TolC multi-drug efflux pump decreases its activity and inhibitory activity against this strain provides evidence whether or not drug-efflux activity possessed by Gram-negative bacteria reduces the inhibitory activity of the extract [16 (link)]. Lipopolysaccharides (LPS) from E. coli were purchased from Sigma-Aldrich. A tetrazolium dye (WST-8) cell viability assay kit (CCK-8) was purchased from Dojindo Laboratories and an ELISA kit for TNF-α was purchased from eBioscience. All measurements from proliferation assays and ELISA experiments were performed using a 96-well microplate reader (Thermo-Fisher, Inc., Waltham, MA, USA). Two-way analysis of variance was performed using four replicate measurements of cell proliferation for each control and treatment group and three replicate ELISA measurements for each control and treatment group.