Dimethyl sulfoxide (DMSO) is currently used seeing that an alternative treatment for various inflammatory conditions as well as for cancer. of the IL-6 level brought on by non-DMSO-treated blood samples in the absence (0%) or presence (100%) of or HSV-1. PGE2 in plasma was quantified using a PGE2 EIA kit according to the manufacturers instructions. Luminex analyses to simultaneously quantify the levels of IL-1, G-CSF, IL-10, IL-13, IL-6, IL-17, MIP-1 (CCL3), VEGF, IFN, IL-12p70, IFN, IL-1RA, TNF-, IL-4 and IL-8 (CXCL8) were performed using a Luminex 100 Bio-Plex Microplate Analyzer (Bio-Rad Laboratories, Hercules, CA). Acquired fluorescence was analyzed by the Bio-Plex Manager? version 6.0 (Bio-Rad Laboratories). DMSO, DMS and DMSO2 To compare the relative efficacy of DMSO with its metabolites, DMS and DMSO2, on modulating inflammatory responses, the 3 compounds were analyzed in the whole blood assay as described above. The three compounds were dissolved in autologous plasma instead of PBS to increase the solubility of DMS in the whole blood assay. Also, to prevent the volatile DMS from affecting the evaluation of other treatments (DMSO and DMSO2), the 96 well plate was sealed with a plate sealer during the 7 h incubation period. Cell Viability Assays In parallel studies, whole human blood samples (70 L/well) were collected at the end of the 7 h incubation period to determine the effect of DMSO, DMSO2 and DMS on cell viability using propidium iodide staining. Specifically, following the 7 h incubation assay, red blood cells were lysed by the addition of 2.5 mL of ammonium chloride solution (StemCell Technologies) in polystyrene flow tubes. After 15 min of incubation on ice, cells were pelleted by centrifugation at 335 x for 5 min, followed by a wash step with cold PBS made up of 2% FBS and 0.05% sodium azide (HFN). Cells were then stained with 1 g/mL propidium iodide in 300 L of HFN and subjected to flow cytometric analysis using 607737-87-1 supplier a FACSCalibur (Becton Dickinson). Fractionation of 607737-87-1 supplier Blood Cells Cells were fractionated from whole blood by Ficoll density gradient centrifugation. Neutrophils were recovered from the granulocyte layer, which was subjected to ammonium chloride lysis to remove red blood cells. Peripheral blood mononuclear cells (PBMCs) were collected from the buffy coat, reconstituted in 50% autologous plasma and seeded 607737-87-1 supplier at 4.5×104 cells/50 L into flat bottom 96 well plates. Monocytes were obtained from PBMCs by adherence of the cell mixture to a flat bottom 96 well plate for 1h in a 37C incubator. The non-adhering lymphocytes were collected and seeded in 50% autologous plasma in individual wells. The monocytes, lymphocytes and neutrophils were then challenged with as described above. After 7 h of incubation, the plates were centrifuged as above and the supernatants collected for IL-6 analysis. Cell Signaling in Human Monocytes White blood cells collected by apheresis from G-CSF-mobilized normal stem cell donors were obtained from the Stem Cell Assay Laboratory/Hematology Cell Bank of the British Columbia Cancer Agency. Monocytes from these apheresis samples were then isolated using an EasySep kit according to the manufacturers instructions and assessed as >92% CD14+ Rabbit Polyclonal to Glucokinase Regulator by flow cytometry or by adherence. They were seeded at 2 x 106 cells/well in flat bottom 12 well plates in serum-free RPMI 1640 medium. After 2 h at 37C, the serum-free medium was removed and 1 mL RPMI 1640 medium DMSO (at a final concentration of 2%) was added to the cells. After 15 min at 37C, the cells were challenged with (at a final concentration 2×105/mL) for 15 and 30 min. Whole cell lysates were prepared for Western blotting by washing cells once with cold PBS followed by the addition of SDS sample buffer (1x) to the cell pellets. The samples were sheared with a 26G needle prior to boiling for 1 min. Whole cell lysates in SDS sample buffer were loaded onto 10% polyacrylamide gels. Upon transfer to PVDF membranes, separated proteins were probed for p-Akt, p-p38, p-JNK, p-ERK1/2 and Grb-2. Primary antibodies were used at 1/1000 dilution, and Grb-2 was used as a loading control. To establish the importance of specific cell signaling pathways to the production of pro-inflammatory cytokines from LPS-stimulated human monocytes, the NF-B inhibitor, Bay11, the p38 inhibitor, SB203580, the PI3K inhibitor, LY294002, and 607737-87-1 supplier the JNK inhibitor, SP 600125, were added to adherent human monocytes cultured in RPMI 1640 made up of 10% AB+ serum in flat bottom 96 well plates. After 15 min at 37C, the cells were challenged with (final concentration 105/mL) for 7 h. Supernatants were recovered and IL-6 levels decided by ELISA 607737-87-1 supplier as above. Differentiation of W16/F10 Cells Mouse melanoma W16/F10 cells were.