Macs magnetic ls separation columns

After islet isolation by the Clinical Islet Transplantation Unit (Mass General Hospital), islet-depleted human pancreatic tissue was dispersed and incubated with CA19-9 and purified using MACS magnetic LS separation columns (Miltenyi Biotec) as described previously (Yatoh et al., 2007 (link)). De-identified organ donors (34–70 years) were non-diabetic and had research consent; exempt status was approved by the Joslin Institutional Review Board.

Dispersed islet-depleted pancreatic tissue remaining after collagenase digestion for islet isolation (Gotoh et al., 1985 (link)) from adult mice and rats was collected from pools of 10–15 mice/rat per sample and divided into 50 mL conical tubes and suspended in RPMI 1640 medium (Invitrogen) supplemented with 10 μm EZSolution Y-27632 ROCK Inhibitor (BioVis) and RNasin Plus RNase Inhibitor (Promega, 100 μL:100 mL). After suspension, tissue was allowed to settle for 5 min, and the supernatant was aspirated to remove low-density components including dead cells and islets. For cell dispersion, 2 mL of 0.25% trypsin/EDTA solution (Invitrogen) and 20 mL PBS (Mg/Ca free) were added, vortexed for several seconds and incubated for 4 min at 37°C on 200 rpm in an incubator shaker. Cold RPMI 1640 medium was added to halt the enzymatic reaction. Following multiple washes and vortexing, tissue was filtered through 40 μm cell strainers (Falcon) to remove clumps. Dissociation into single cells was confirmed by light microscopy. Most acinar tissue (larger cells) was not viable at this stage, as assessed by trypan blue exclusion. Duct cells were then purified using MACS magnetic LS separation columns (Miltenyi Biotec) according to the manufacturer's instructions with anti-CD24 and/or anti-CD44 antibodies (Table S4 for antibodies).