F4 80 magnetic beads

Pancreata from 8 week old, non-transgenic mice were harvested by washing the pancreas in HBSS, mincing the tissue, centrifuging (931 × g, 4°C, 2 min), and then dissociating the tissue in collagenase (2 mg/ml, 5 ml, 37°C, 20 min, 220 rpm). To stop dissociation, HBSS + 5% FBS was added to the pancreas–collagenase mixture and washed two additional times with HBSS + 5% FBS (931 × g, 4°C, 2 min). Pancreas cells were then filtered through 500 μm and 105 μm meshes prior to adding the cell suspension to HBSS + 30% FBS and centrifuging (233 × g, 4°C, 2 min) to obtain a cell pellet. Cells were then labeled with F4/80 magnetic beads (Miltenyi Biotec, Auburn, CA) to isolate pancreas-resident macrophages. Cells were filtered through a 40 μm mesh to obtain a single-cell suspension, and then cells were incubated with F4/80 magnetic beads in MACS buffer (15 min, 4°C, as per the manufacturer’s instructions) (MACS buffer: PBS, 0.5% BSA, 2 mM EDTA). Cells were washed with MACS buffer (300 × g, 4°C, 10 min), filtered through 40 μm mesh, and then applied to pre-washed LS Columns (Miltenyi Biotec, Auburn, CA). Per column instructions, magnetically labeled cells were acquired and plated in DMEM-F12 + 10% FBS + 1% l-glutamine + penicillin/streptomycin + 0.1 μg/ml macrophage colony stimulating factor (Peprotech, Rocky Hill, NJ).

Liver cells from Alk1^fl/fl Clec4f^Cre mice were first isolated by F4/80 magnetic beads (Miltenyi Biotec) and then sorted by FACS. Genomic DNA was extracted using the QIAamp DNA Micro Kit (QIAGEN). PCR was performed as follows: 95°C for 3 minutes, 35 cycles at 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 45 seconds, followed by a 2-minute incubation at 72°C. PCR primers for floxed Alk1 were as follows: forward, 5′-GCTTGCATGCTTGGCTCTAC-3′; reverse, 5′-GGGAGGAGCCATGTTCTCAG-3′. PCR primers for Alk1 deletion were as follows: forward, 5′-GTGGCTGGAGAGGAACAGTAGTCC-3′; reverse, 5′-TGGAGACCTGCTCTGAGATGTCTG-3′. (See complete unedited blots int the supplemental material.)