Biocoll

PBMCs were isolated using density gradient centrifugation by layering blood onto Biocoll (Millipore) and centrifugation at 1200×g for 20 min without brake. The resulting buffy layer was then washed twice with PBS before use in subsequent experiments. Monocyte enrichment was performed using the RosetteSep™ Human Monocyte Enrichment Cocktail (Stemcell Technologies). Positive enrichment of CD14⁺ monocytes was performed using CD14 Magnetic Particles (Becton–Dickinson). Cells were counted using trypan blue staining on a Neubauer hemocytometer and cultured at 37 °C with 5 % CO₂, in RPMI 1640 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine (all Biochrom), and 10 % AB serum.

Pancreatic islets were isolated via bile duct collagenase digestion (Collagenase P, Sigma) and Biocoll (Millipore) gradient separation and left to recover overnight at 37°C in RPMI 1640 with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. For insulin release assays, five equally sized islets per mouse (in duplicate) were statically incubated in Krebs-Ringer buffer the following morning (∼ZT6) and stimulated for 1 h at 37°C with 2 or 20 mM glucose or 30 mM KCl. Supernatant was collected and assayed for insulin content by ELISA. Islets were then sonicated in acid-ethanol solution and solubilized overnight at 4°C before assaying total insulin content by ELISA. Percent insulin content secreted as a response to stimulus was normalized to basal secretion levels for each condition and reported as fold change from control. For insulin release assays in cell lines, 3-4 × 10⁶ cells were cultured for 3 d in 6-cm suspension culture dishes. Formed pseudoislets were then transferred into new suspension culture media and left to recover overnight. Glucose-responsive insulin secretion was performed as described above, using 10 pseudoislets per sample and a basal glucose level of 0 mM glucose instead of 2 mM.

CD14⁺ cells were isolated from buffy coats from healthy donors obtained from Red Cross Blood Service, Baden-Württemberg. In brief, after a gradient centrifugation using Biocoll (Merck Millipore), monocytes were isolated by magnetic-activated cell sorting via labeling with anti-CD14 microbeads (Miltenyi). 1x 10⁶ cells/mL were seeded in X-VIVO™ 15 medium (Lonza) for up to seven days at 37°C and 7.5% CO₂. pBM were stimulated with M-CSF (100 ng/mL, Peprotech), IL-4 (10 ng/mL, Peprotech), dexamethasone (1x 10^-7 M; 1000 U/mL, Sigma-Aldrich, St. Louis, MO, USA), GM-CSF (100 ng/mL, Peprotech), IFN-γ (10 ng/mL), IL-1β (10 ng/mL, Peprotech) and LPS (1 μg/mL, Invitrogen) as indicated.