Therefore, the defect in ovalbumin (OVA) -specific IgA production is unlikely to be linked to the reduced frequency of CD11b+ DC but rather would be linked to the lack of CD47 expression by non-haematopoietic

cells. CD47−/− BALB/c (back-crossed for 16 generations) and DO11.10 mice were bred in specific pathogen-free conditions at the Experimental Biomedicine Animal Facility, University of Gothenburg. BALB/c (WT) mice were purchased from Taconic, Ry, Denmark. To generate bone marrow (BM) chimeric mice, BM cells from donor WT mice were filtered, red blood cells were lysed, and the remaining cells Ibrutinib were resuspended in PBS. Recipient WT or CD47−/− mice were irradiated (1000 rad) before 2 × 106 to 5 × 106 donor BM cells were transferred intravenously to generate WT  CD47−/− (WT/CD47) chimeras or CD47−/−  CD47−/− irradiation controls (CD47/CD47) and WT  WT (WT/WT). Irradiated mice and mice which underwent mesenteric lymphadenectomy were left to recover for 6 weeks before being included in experiments. The

chimerism was confirmed by flow cytometry. All experiments performed SCH772984 were approved by the Swedish government’s Animal Ethics Committee and followed institutional animal use and care guidelines. Cells were isolated from LN and spleen by mechanical disruption. For DC isolation, tissues were pre-treated with liberase (0·4 mg/ml; Roche, Indianapolis, IN) in Hank’s buffered saline solution (HBSS, GIBCO/Invitrogen, Leek, The Netherlands) supplemented with 2% fetal bovine serum (FBS) FER at 37° for 30 min. Small intestines were flushed with calcium-free and magnesium-free HBSS (GIBCO/Invitrogen) and cut into smaller pieces. The PP were excised from intestinal tissue and washed. For removal of epithelial cells, tissues were incubated at 37° for 15 min with HBSS containing EDTA (5 mm),

FBS (2%) and antibiotics, and then shaken vigorously. The procedure was repeated twice for small intestinal lamina propria (LP) and once for PP. The LP was then digested with collagenase D (100 U/ml; Roche) in RPMI-1640 medium supplemented with FBS (10%), HEPES (15 mm) and antibiotics during two 1 hr incubations. The PP were digested with liberase (0·4 mg/ml) in HBSS containing polymycin B (10 U/ml) at 37° for 27 min. Remaining tissue was disrupted over nylon mesh and counted using a cell counter (Sysmex, Kungsbacka, Sweden) or manually using trypan blue to exclude dead cells. Mesenteric lymph nodes and small intestines were frozen in OCT compound, then 8-μm cryosections were collected on gelatin-coated slides, air-dried and fixed in 1% paraformaldehyde for 5 min.