All of the experimental procedures were in accordance

All of the experimental procedures were in accordance selleck inhibitor with institutional, state and federal guidelines on animal welfare and every effort was made to minimize suffering. The animal experiments were approved by the Regierungspr?sidium Freiburg and supervised by the Animal Protection Representatives of the University Freiburg Medical Center or the MPI. Mice were anesthetized before sacrificing with 1% pelltobarbitalum natricum at the dose of 10 mg/kg. BMDC were prepared from bone marrow cells obtained from the femur and tibia in RPMI 1640 (Lonza, Basel, Switzerland) and 10% FBS (Cambrex), supplemented with 20 ng/ml GM-CSF from a GM-CSF expressing line. Cells (1��106 cells/ml) were washed and recultured with fresh medium containing 20 ng/ml GM-CSF every 3 d for 8 d. BMDC were cultured with PDWGF(100 ��g/ml) for 21.

5 h, and then 2 mM ATP (Sigma) was added for an additional 2.5 h. When indicated, Z-YVAD-fmk (10 ��M) was added 30 min before PDWGF stimulation. Cells were used for flow cytometry analysis, or cell lysates were prepared and analyzed by western blot. Cell culture supernatants were analyzed by ELISA. ELISA The concentrations of human IL-1��, IL-1��, IL-18 and TNF-�� as well as murine IL-1�� and TNF-�� were measured by commercial ELISA Duo Set Kits (R&D Systems) or ELISA MAX kits (Biolegend) according to manufacturer instructions. FLICA Staining Active caspase-1 was detected using the FLICA caspase-1 assay kit. Briefly, human PBMC (0.5��106 cells/0.5 ml) were treated with PDWGF (100 ��g/ml) for 16 h prior to treatment with fluorescein-labeled inhibitor Z-YVAD-fmk (10 ��M) for 1 h at 37��C.

Cells were washed three times and analyzed by flow cytometry on a FACSCalibur (BD Biosciences), and the data were analyzed using CellQuest software (BD Biosciences). Flow Cytometry BMDC exposed to PDWGF, LPS, or OVA were stained with the relevant mAbs or isotype controls in a FACS bufer for 30 min on ice. In order to reduce non-specific Fc receptor-mediated binding, Fc block (CD16/CD32) from BD Biosciences was added to cells prior to and during staining. Drug_discovery Cells were acquired on an LSR II flow cytometer (BD Biosciences) and DCs gated according to the FSC, SSC, and CD11c+ parameters for analysis. Western Blotting After treatment with Z-YVAD-fmk (10 ��M), quinidine (100 ��M), glybenclamide (100 ��M), KN-62 (10 ��M), KCl (50 mM), NAC (30 mM), TPCK (25 ��M), SP600125 (10 ��M), SB203580 (20 ��M), and UO125 (10 ��M) for 30 min, cells (1��106) were stimulated with PDWGF (100 ��g/ml) for 24 h. Cell supernatants were collected and cell lysates were prepared as previously described [24].

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