Then the labeled cells were washed and incubated with anti-FITC-conjugated magnetic beads (Miltenyi Biotec). Positive cells were sorted using columns and a MACS kit (Miltenyi Biotec). Finally, the purities were tested (>90%). The purified γδ T cells were stimulated

with either IL-1β or IL-23 or the combination for 48 hours. The supernatants were collected for measurement of IL-17A. The remaining cells were directly stained for intracellular IL-17A either without additional stimulation or with phorbol-12-myristate-13-acetate (PMA, 50 ng/mL; Sigma-Aldrich), ionomycin (1 μg/mL; Sigma-Aldrich), and monensin (5 μg/mL; Sigma-Aldrich) for 5 hours. To measure IL-23 secretion by macrophages stimulated with HMGB1, peritoneal macrophages were harvested from TLR4+/+ mice or TLR4−/− mice 3 days after treatment with Linsitinib 3% sodium thioglycolate. The cells were stimulated with HMGB1 (20 ng/mL, eBioscience) for 18 hours and the supernatant was see more collected for IL-23 measurement. The concentrations of IL-17A, IL-23, IL-23p40, and HMGB1 were measured by a standard enzyme-linked immunosorbent

assay (ELISA). The following ELISA kits were used: IL-17A and IL-23p40 (Dakewe Biotech, Shenzhen, China); IL-23 (Biolegend, USA); and HMGB1 (Yanhui Biotech, Shanghai, China). To isolate hepatic leukocytes, livers were pressed through a 200G stainless steel mesh and suspended in PBS. The suspension was centrifuged at 50g for 1 minute. PDK4 The supernatant was then transferred into a new tube and centrifuged at 800g for 10 minutes. The pellets were resuspended in 40% Percoll and centrifuged at 1,260g for 15 minutes at room temperature. The pellets were resuspended and the cell number was determined. To detect hepatic neutrophils, 1 × 106 cells were stained with specific mAb against mouse FITC-CD11b (M1/70, BD Bioscience, USA), PE-Ly6G (1A8, BD Bioscience), Percp-Cy5.5-CD45.2 (104, BD Bioscience), and APC-Gr-1 (RB6-8C5, BD Bioscience). To detect

γδ T cells, 1 × 106 cells were stained with specific mAb against mouse FITC-γδTCR (GL3, eBioscience), PE-CD3 (145-2C11, BD Bioscience), and Percp-Cy5.5-CD45.2 (104, BD Bioscience). To detect IL-17A+ cells, 1 × 106 cells were stimulated with PMA (50 ng/mL), ionomycin (1 μg/mL), and monensin (5 μg/mL) for 4 hours. The cells were stained with FITC-CD4 (RM4-5, BD Bioscience), Percp-Cy5.5-CD3 (145-2C11, BD Bioscience), APC-γδTCR (GL3, eBioscience), and PE-CY7-NK1.1 (PK136, BD Bioscience), and then intracellularly stained with PE-IL-17A (BD Bioscience) after fixation and permeabilization. Finally, the stained cells were analyzed using a FACSCalibur (BD Biosciences) or BD LSR II (BD Biosciences) flow cytometer. The acquired data were analyzed using FlowJo software. Data are presented as the mean ± standard error of the mean (SEM). The significance of differences was determined using a two-tailed unpaired t test; the significance levels are marked *P < 0.05; **P < 0.01; ***P < 0.005.