Fluorescence-activated cell sorting analysis was performed using FACScalibur and/or FACSverse (both from Becton Ridaforolimus nmr Dickinson). Mice were injected with IL-25 and D-Gal/LPS, as described above, and HMNCs were isolated 8 hours later. Cells were blocked using Fc-block and stained using the following monoclonal antimouse Abs: APC-Cy7 anti-Ly6G; PE anti-Ly6C; V450 anti-GR1
(all from Becton Dickinson); and PerCP anti-CD11b (BioLegend, San Diego, CA, USA). CD11b+GR1+ cells as well as CD11b+Ly6GhighLy-6C+ and CD11b+Ly6G-Ly-6Chigh subsets were sorted using FACSAria II (Becton Dickinson). Purity of sorted cells was >90%, as evaluated by FCM. T cells were purified from spleen of BALB/c mice by magnetic cell separation. Briefly, splenocytes were passed through a 70-μm nylon mesh cell strainer, and T cells were isolated using a CD90.2+ cell isolation kit (Miltenyi Biotec), according to the manufacturer’s instruction. The resulting cell preparations contained more than 95% CD3+ T Erismodegib clinical trial cells, as assessed by FCM. T-cell proliferation was assessed using carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes,
Eugene, OR). Purified CD11b+GR1+ cells as well as CD11b+Ly6GhighLy-6C+ or CD11b+Ly6G-Ly-6C+ subsets were cultured at different ratios with syngenic purified CFSE-positive T cells stimulated with anti-CD3/CD28-activating Abs (MiltenyiBiotec). Coculture was performed in a 96-well plate in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin (all from Lonza, Milan, Italy). T-cell proliferation was determined after 72 hours of culture by FCM. RNA was extracted from GR1/CD11b+ and GR1/CD11b− cells isolated from livers of mice injected with IL-25 and D-Gal/LPS, as described above, and used for inducible nitric oxide synthase (iNOS) and arginase
II RNA expression by real-time polymerase chain reaction (PCR; see Supporting Materials). Mice were given IP a depleting antimouse GR1 Ab (250 µg/mouse) or control IgG (250 µg/mouse; both from R&D Systems) 36 hours before IL-25 administration. Blood samples were collected selleck 6 hours after D-Gal/LPS hepatitis induction by retro-orbital bleeding, and sera were stored at −80°C. Mice were euthanized 2 hours later, and livers were explanted for RNA and protein extraction, isolation of mononuclear cells, evaluation of GR-1 cell depletion, and histopathological analysis. Please see the Supporting Materials for details on terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Please see the Supporting Materials for details on real-time PCR. Please see the Supporting Materials for details on protein extraction, western blotting and enzyme-linked immunosorbent assay (ELISA). Please see the Supporting Materials for details on histopathological analysis and immunofluorecence. Please see the Supporting Materials for details on AML12 cell culture and apoptosis assay.