NOD/LTj see more mice were bred in our own facility under specified pathogen-free conditions. Breedings were done from the age of 8 weeks and older. The appearance of the vaginal plug was noted as E0.5. Pregnant mice were sacrificed and embryos dissected at embryonic age of E15.5. BM cells were isolated from the femora from mice of 8 weeks. All mice were female and were supplied with water and standard chow ad libitum. Experimental procedures were approved by the Erasmus University Animal Ethical Committee.
Embryonic (E15.5) pancreas (pooled) and liver were isolated and micro-dissected from the stomach and digested with Collagenase Type 1 (1 mg/mL), hyaluronidase (2 mg/mL) (both Sigma Aldrich, St. Louis, MO, USA)
and DNAse I (0.3 mg/mL) (Roche Diagnostics, Almere, The Netherlands) for 10 min at 37°C. Embryonic pancreas and liver cells were flushed through a 70 μm filter and washed. Pancreases of 5-week-old mice were isolated after a cardiac perfusion and cut into small pieces and digested with Collagenase Type 1, hyaluronidase and DNAse I for 40 min at 37°C. Cells were flushed through a 70 μm filter and washed. Blood of 4 week old mice was collected selleck chemical in EDTA tubes using a heartpunction. Erythrocytes were lysed with NHCL2 buffer and washed. Single-cell suspensions of BM were prepared as described previously 39. All cells were resuspended in PBS containing 0.1% BSA and were ready for flow cytometry staining. Single-cell suspensions from pancreas (E15.5 and 5 wk) were labeled with mAbs. Ceramide glucosyltransferase Antibodies used were ER-MP58-biotin (own culture), Ly6C-FITC (Abcam, Cambridge, UK), Ly6G-Pacific Blue (Biolegend, Uithoorn, The Netherlands), CD11b-allophycocyanin-Cy7,
CD86-PE (both Becton Dickinson, San Diego, CA, USA), CD11c-allophycocyanin, CD11c-PE, CD11c-PE-Cy7, CD86-Pacific Orange, F4/80-PE-Cy5 (all eBiosciences, San Diego, CA, USA), MHC class II-PE (C57BL/6, clone M5/114, Becton Dickinson) and MHC class II-biotin (NOD clone 10.2.16, own culture). Afterwards cells were washed and incubated with streptavidin-allophycocyanin (Becton Dickinson). To detect proliferation, the cells were fixed in 2% paraformaldehyde, and permeabilized using 0.5% saponin. Subsequently, cells were incubated with Ki-67-FITC (Becton Dickinson) diluted in 0.5% saponin, washed and resuspended in 0.1% BSA. Cells suspensions were analyzed using a FACS Canto HTSII (Becton Dickinson) flow cytometer and FACS Diva and Flowjo software. Antigen processing was determined by measurement of the fluorescence upon proteolytic degradation of the self-quenched conjugate DQ-Ovalbumin 40. Briefly, cells were resuspended in PBS with 2% FCS and 100 μg/mL DQ-Ovalbumin (Molecular Probes, Breda, The Netherlands) and incubated for 30 min at 37°C.