Vorinostat saha

HCEC (Human Colon Epithelial Cells) were obtained from Nestlé Ltd. Research Center (Lausanne, Switzerland). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 2 mM L-glutamine supplemented with 10% fetal bovine serum, 45 IU/ml penicillin, 45 IU/ml streptomycin, 9.11 µl/ml sodium pyruvate, 4.9 × 10^–3 µl/ml phosphoethanolamine, 4.9 × 10^–3 µl/ml ethanolamine, and 3 mg/ml BSA until 90–95% confluency before being subcultivated. For each experiment, 1 × 10⁶ cells were seeded on 150 mm diameter dishes. After 24 h, when cells were in the exponential phase of growth, they were stimulated with 1 µM Vorinostat (SAHA, Sigma-Aldrich, Steinheim, Germany), or 30 ng/ml (~ 0,1 µM) Trichostatin A (TSA, Sigma-Aldrich, Steinheim, Germany) for the indicated time period. For the last 24 h of the incubation TNFα (20 ng/ml) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or vehicle was added. Vehicle controls were performed for every time point. Cultivation of cells was carried out in a humidified incubator at 37 °C with 5% CO₂ in O₂. Before use, the cells were tested for mycoplasma contamination.

Whole blood was obtained from healthy volunteers. Ethical approval was granted by South Sheffield Regional Ethics committee (07/Q2305/7). Peripheral blood mononuclear cells were separated by differential centrifugation using a Ficoll-Paque gradient and differentiated into monocyte derived macrophages (MDM) for 14 d as previously described in 24 well plates (Corning) (26 (link)). Bacteria were washed in PBS and re-suspended in RPMI 1640 supplemented with 10% pooled human immune serum (from previously vaccinated volunteers with demonstrable antibody levels to serotype 2 pneumococci) (27 (link)). MDM were challenged with either opsonised S. pneumoniae, Δply or PBS, at a MOI of 10, rested on ice for 1 h and incubated at 37°C in 5% CO₂ for a further 3 h (28 (link)). For certain experiments cells were treated with 3 μM vorinostat (SAHA, Sigma) or 0.5% DMSO (vehicle control) for 30 min prior to bacterial challenge and vorinostat reintroduced after bacterial challenge.