In vitro treatment of B-1 B cells with hapten–protein Selleckchem Gemcitabine complex.  Naïve wild-type B-1 B cell-containing

peritoneal cells were incubated with the hapten–protein complex trinitrophenyl–bovine serum albumin (TNP–BSA, prepared at a concentration of 50 μg/ml in RPMI 1640 culture media supplemented with 5% FCS) for 40 min at 37 °C. Incubation of iNKT cells with B-1 B cells.  B-1 B cell-containing peritoneal cells exposed to TNP-BSA and iNKT cell-containing LMNC exposed to lipid extracts were washed and co-incubated in vitro for 40 min at 37 °C. Centrifuged pellets of the activated iNKT and B-1 B cell mixture were resuspended in PBS prior to adoptive transfer. Adoptive transfer.  To reconstitute iNKT cells in Jα18−/− or CD1d−/− mice, we transferred LMNC into Jα18−/− or CD1d−/− mice at a dose of 0.5–1 × 106 cells per mouse. To reconstitute B-1 B cells, we transferred the mixture of peritoneal cells and LMNC into JH−/− or CBA/N-xid mice at a dose of 5 × 106 cells per mouse. Cells were transferred via intravenous injection into the retro-orbital plexus of recipient mice under methoxyflurane anaesthesia 1 day prior to challenge (i.e., day 3 after sensitization). Flow cytometry with CD1d-α-GalCer Selleck JNK inhibitor tetramers.  Liver mononuclear cells were washed and resuspended in PBS staining buffer containing 2% BSA, stained with a mixture of FITC-anti-TCR-β antibody and PE-labelled CD1d-α-GalCer

tetramers on ice for 30 min and washed twice more. The double-positive cells (iNKT cells) were identified using a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and reported as a percentage of total αβ-TCR-positive LMNC (T cells). iNKT cells constitute

approximately 70% of hepatic T cells in the wild-type H-2d mice employed here. Results were analysed using Mac CellQuest (BD). Isolation and flow cytometry of for hepatocytes.  Mice were anesthetized with intra-peritoneal pentobarbital before entering the abdomen. Portal veins were perfused with Hanks A solution for 3–4 min and Hanks B solution with collagenase until signs of liver digestion became apparent. The livers then were removed. The hepatocyte fraction was strained through a 70-m mesh (BD) and stained with FITC-anti-CD1d antibody for 1 h on ice before analysis by flow cytometry. Results were analysed using Mac CellQuest (BD). It is not understood how iNKT cells respond so rapidly to contact sensitization. Our hypothesis was that the character of hepatic lipids changes in a manner that increases their capacity to stimulate iNKT cells. To investigate this, we utilized adoptive transfer techniques in JH−/− and CBA/N-xid mice, which lack B cells and B-1 B cells, respectively. Both strains thus have impaired CS at baseline at both 2 and 24 h after challenge (Group B in Fig. 1A,B). We previously demonstrated that CS is impaired in these B cell–deficient mice compared with wild-type mice and that CS could be fully reconstituted with adoptive transfer of sorted B-1 B cells previously activated in vivo [8, 10].