Mifn γ

IL-22ra1^ΔβKO and their wildtype littermates (IL-22ra1^fl/fl) were euthanised and freshly prepared collagenase P (Roche, Indianapolis, IN) solution (0.5 mg mL⁻¹) was injected into the pancreas via the common bile duct. The perfused pancreas was then digested at 37 °C for 20 min, and islets were purified via centrifugation on a Histopaque-1077 (Sigma; #10771) gradient. Islets were then handpicked under a stereoscopic microscope, enumerated, and cultured overnight in 5 mM glucose.
Following overnight culture, human islets were plated at 10 islets per well in 5.5 mM glucose. We conducted static glucose-stimulated insulin secretion tests by challenging islets (mouse and human) consecutively with 2.8 mM glucose for 30 min, 20 mM glucose for 30 min and 100 nM GLP-1 (7–36 active peptide; Sigma-Aldrich) in 20 mM glucose for a further 30 min. We collected supernatants for insulin and proinsulin secretion measurements, and the islets were homogenized in Trizol for RNA analysis. A subset of wildtype islets was also stimulated with 10 ng mL⁻¹ mIFNγ (R&D; Cat # 485-MI) in the presence/absence of 50 ng mL⁻¹ mIL-22-Fc for 48 h.

For in vitro differentiation of monocytes into macrophages, FACS-sorted cells were suspended at 0.5×10⁶ cells/mL in RPMI 1640 medium supplemented with 10% HI-FCS and 1% PenStrep (10378016; Gibco). CD14⁺HLA-DR^neg/low/CD14⁺HLA-DR^high monocytes were cultured in 96 well plates (200 µL/well) in the presence of 20 ng/mL M-CSF (216-MC-005; R and D Systems) for 4 days (Murray et al., 2014 (link)). Monocyte-derived macrophages [(Mb), in RPMI 1640 medium supplemented with 2% HI-FCS] were stimulated with IFN-γ [M(IFN-γ), 20 ng/mL; 285-IF; R and D Systems], IL-4 [M(IL-4), 20 ng/mL, 130-093-920; Miltenyi Biotec], or dexamethasone [M(dexa), 1 µM; Sigma] (Xue et al., 2014 (link)) for 48 hr.