Cd45 beads

Pancreases of 5 week old mice were isolated after a cardiac perfusion, cut into small pieces 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 40 minutes at 37°C. Subsequently, cells were flushed through a 70 µm filter and washed with DMEM +10% FCS. All cells were resuspended in PBS containing 0.1% BSA and were ready for flow cytometric staining.
Single-cell suspensions from pancreas were labeled with CD45 beads (Miltenyi, Leiden, The Netherlands) and CD45⁺ cells were pre-sorted with the AutoMACS pro (Miltenyi) to remove most of the non-immune cells. The pancreatic CD45⁺ cells were further processed for FACS analysis or DC isolation.

Single-cell suspensions were performed from the intestine by mechanical disruption and passage through filter (Miltenyi Biotec). Lamina propria cells and intraepithelial cells were isolated using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to manufacturer’s instructions. In short, tissues were transferred to preheated digestion solution in C tubes (Miltenyi Biotec) and processed by gentleMACS Octo Dissociator (Miltenyi Biotec). The obtained cell suspension was filtered on a 100 μm cell strainer and washed with MACS buffer. Cells were centrifuged (300 × g, 4 °C, 10 min) and counted using the TC20 automated cell counter (Bio-Rad). Leukocytes were depleted or isolated by incubating the cell suspension with CD45 beads (Miltenyi Biotec). For endothelial activation, the CD45-negative lamina propria cells were further isolated using CD31 beads (Miltenyi Biotec).