Belinostat pxd101

To estimate NETosis kinetics, we used a cell-impermeable DNA binding dye called Sytox Green (Life Technologies, Carlsbad, CA, USA). A concentration of 5 µM Sytox Green were added in 100 µL media (RPMI 1640 medium supplemented with 10 mM HEPES) containing 50,000 neutrophils and then seeded into 96-well black clear-bottom plates. A volume of 5 µL of HDACis (final concentrations: 0.5, 1, 2, 4, 5, 10, 20 and 40 µM belinostat (PXD-101, Selleckchem); 0.08, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 µM panobinostat (LBH589, Selleckchem) was added to respective wells with controls (RMPI + neutrophils only) for 30 min at 37 °C and 5% (v/v) CO₂. A volume of 5 µL agonists (final concentrations: 25 nM PMA; 4 μM A23187; 5 μg/mL LPS (from E. coli 0128); 5 μM ionomycin, (unless otherwise stated)) was then added and placed at 37 °C and 5% (v/v) CO₂ incubator. By using a fluorescence plate reader, the Sytox Green fluorescence intensities were measured every 60 min for up to 4 h (504 nm excitation, 523 nm emission, POLARstar OMEGA, BMG Labtech, Guelph, ON, Canada). Finally, the NETotic index (% of Sytox Green accessible total DNA) was calculated by subtracting the baseline green fluorescence at time 0-min from the fluorescence at each time point and was then divided by the fluorescence values of 100% NET formation obtained by lysing the cells with 0.5% (v/v) Triton X-100 (240 min time point).

Cell samples were treated with 1 μM Retinoic acid (Sigma-Aldrich, St. Louis, MO, USA), 2 nM or 8 nM Idarubicin (Sigma-Aldrich), 0.5 μM 3-Deazaneplanocin A (Cayman Chemical Company, Ann Arbour, MI, USA), and 0.2 μM Belinostat (PXD101) (Selleckchem, Munich, Germany) in different combinations. Cell proliferation and survival were evaluated by trypan blue exclusion test. Cells were mixed with 0.2% of trypan blue dye (final concentration). Viable and dead (blue coloured) cell numbers were determined by counting the cells in a haemocytometer under the light microscope. For the detection of apoptosis, we used the assay “ApoFlowEx® FITC Kit” (Exbio, Vestec, Czech Republic) according to the manufacturer's instructions. This assay detects viable, early apoptotic, and late apoptotic or necrotic cells according to how they get stained by Annexin V-FITC and Propidium Iodide. Stained cells were analysed on the BD FACS Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Cell cycle distribution was analysed using standard propidium iodide staining procedure [15 (link)].

Gelatin, putrescine, selenium, progesterone, apotransferrin, glucose and insulin were obtained from Sigma (Steinheim, Germany). Accutase was obtained from PAA (Pasching, Austria). FGF-2 (basic fibroblast growth factor), noggin and sonic hedgehog were obtained from R&D Systems (Minneapolis, MN, USA). Y-27632, SB-43154 and dorsomorphin dihydrochloride were obtained from Tocris Bioscience (Bristol, UK). Matrigel^TM was obtained from BD Biosciences (Massachusetts, USA). All cell culture reagents were obtained from Gibco/Invitrogen (Darmstadt, Germany), unless otherwise specified. The following chemicals (HDACis and mercurials) were obtained from Sigma unless otherwise specified. The vehicles used are also mentioned with the compounds: (HDACis) valproic acid (VPA, P4543; water); trichostatin (TSA, T1952; DMSO); vorinostat (SAHA, SML 0061; DMSO); belinostat (PXD101, S1085, Selleckchem; DMSO); panobinostat (LBH589, S1030, Selleckchem; DMSO); entinostat (MS-275, Cay-13284-25; Biomol; DMSO); (mercurials) methylmercury (MeHg, 442,534; 10 % ethanol); thimerosal (THM, T4687; water); mercury(II)chloride (HgCl₂, 203,777; water); mercury(II)bromide (HgBr₂, 437859, water); 4-chloromercuribenzoic acid (PCMB, C5913-5G; water); and phenylmercuric acetate (PMA, P27127-25G).