Livers were perfused with 10 ml of phosphate-buffered

saline (PBS) via the portal vein to remove circulating lymphocytes. Liver and spleen single-cell Selleckchem BIBW2992 suspensions were prepared from whole tissue by mechanical disruption in RPMI-1640/2% (v/v) fetal bovine serum (FBS). Bulk liver non-parenchymal cells (NPC) were enriched by density centrifugation using Histodenz (Sigma, St Louis, MO, USA). B cells were purified by CD19-positive selection using the magnetic affinity cell sorting (MACS) system (Miltenyi Biotec, Auburn, CA, USA). mDCs were purified as described [18]. Briefly, liver and spleen cells were depleted of NK1·1+, CD3+, CD19+ and/or plasmacytoid dendritic cell antigen-1 (PDCA-1)+ cells, followed by positive selection of CD11c+ cells using the MACS system (Miltenyi Biotec). B cells were isolated from wild-type mice 18 h after LPS [100 μg/kg intraperitoneally (i.p.); Alexis Biochemistry, San Diego, CA, USA] or PBS administration. In some experiments, mice were given poly I:C

(4 mg/kg, i.p.) for DAPT mw 18 h. The purity of mDCs and B cells was consistently > 90%. mDCs were isolated from wild-type and B cell-deficient μMT mice given the endogenous DC poietin fms-like tyrosine kinase 3 ligand (Flt3L) (10 μg/mouse/day; i.p. for 10 days; Amgen, Thousand Oaks, CA, USA), with either PBS or LPS (100 μg/kg, i.p.) treatment for the last 18 h. B cell-depleted liver NPCs were stimulated with LPS (10 ug/ml) for 48 h in the presence or absence of liver or spleen B cells. Activation of mDCs was determined by the level of expression of CD80, CD86 and programmed cell death 1 Plasmin ligand 1 (PD-L1) (B7-H1; CD274) on CD19–B220–CD11c+ cells. Single-cell suspensions were blocked for 10–15 min with anti-CD16/32 followed by staining with a fluorescent-tagged antibody mixture

directed against the cell surface markers CD1d, CD3, CD5, CD19, CD23, CD24, CD39, CD40, CD80, CD86, PD-L1, B220, CR1/2, immunoglobulin (Ig)M and IgD (BD PharMingen, Franklin Lakes, NJ, USA or BioLegend, San Diego, CA, USA). Data were acquired on a LSR II or LSR Fortessa (BD Bioscience, San Jose, CA, USA) and analysed with FlowJo software (Tree Star, Ashland, OR, USA). Purified B cells were cultured with or without 500 ng/ml phorbol myristate acetate (PMA), 1 μM ionomycin and 10 μg/ml LPS; purified mDCs were cultured with or without 10 μg/ml LPS. The cells were maintained for 48 h at 37°C in RPMI-1640 supplemented with 50 μM 2-mercaptoethanol (ME), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Supernatants were collected and cytokine production measured using a cytometric bead assay (CBA) Flex Set system (BD Bioscience) and analysed using FCAP Array Software (BD Bioscience). Bulk splenocytes and liver non-parenchymal cells (NPC) were activated for 5 h with 10 μg/ml LPS, 500 ng/ml PMA (Sigma) and 1 μM ionomycin (Sigma) in the presence of GolgiStop (BD Bioscience), followed by staining with fluorescent-labelled CD19 monoclonal antibody (mAb).