Anti mouse il 1β apc monoclonal rat igg2b clone 166931

Flow cytometry analysis of tumor MNCs was performed using directly conjugated monoclonal mouse antibodies: anti-mouse F4/80 Pacific Blue (clone BM8), anti-mouse CD49b PerCP/Cy5.5 (clone DX5), anti-mouse CD11c FITC (clone N418), and anti-mouse CD3 APC/Cy7 (clone 17A2) (Biolegend, San Diego, CA, USA). Surface staining was performed according to the manufacturer’s instructions. For intracellular staining with IL-1β, the MNCs were fixed and permeabilized for 20 min at 4 °C with Cytoperm/Cytofix (BD Biosciences, Franklin Lakes, NJ, USA) and stained with anti-mouse IL-1β APC (Monoclonal Rat IgG2B Clone #166931) (R&D Systems, Minneapolis, MN, USA), antibodies in BD Perm/Wash solution according to the manufacturer’s instructions. Sample analysis was performed on a MACSQuant Analyzer (Miltenyi Biotec) and flow cytometry data were analyzed with FlowJo 8.7. software (FlowJo, LLC, New York, NY, USA).

MSC were plated at a density of 15,000 cells/96-well in complete MSC medium. Twenty-four h later, splenocytes from control mice were added at a ratio of 10 to 1 MSC in 90 µl of RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin with/out 25 ng/ml LPS (Merck). After 2 h, 0 or 10 µl of ATP was added to the LPS conditions at a final conc. of 5 mM, whereas to the other wells 10 µl of PBS was added. After 1 h, cells were fixed and permeabilized for 20 min at 4 °C with Cytoperm/Cytofix (BD Biosciences, Franklin Lakes, NJ, USA) and stained with anti-mouse F4/80 Pacific Blue (BM8), anti-mouse CD49b PerCP/Cy5.5 (DX5), CD11c APC (N418) or CD11c PE (N418) (Biolegend), anti-mouse ASC PE (Biolegend) and anti-mouse IL-1β APC (Monoclonal Rat IgG2B Clone #166931) (R&D Systems) antibodies in BD Perm/Wash solution according to the manufacturer’s instructions. Sample analysis was performed on a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and flow cytometry data were analyzed with FlowJo 8.7. software (FlowJo, LLC, RO, USA).