Isolation and characterization of an islet-associated CD45- MHC class II+ cells using single cell RNA sequencing.
MHC-II is expressed in normal murine and human islets by cells that are located at the periphery of the islet and associated with the efferent vasculature. These cells are non-hematopoietic in origin based on their non-expression of CD45, and split in two families, vascular and fibroblastic, based on their gene transcription profiles, their morphology, and their location. Overall design: Fresh human islets (IIDP, Prodo laboratories) were washed once with PBS prior to digestion with trypsin EDTA (Corning) for 20 minutes on ice with light shaking every 5 minutes68. Islets were gently dissociated with a P1000 pipette prior to stopping the enzymatic reaction with DMEM-10% serum. Cell suspensions were filtered through 0.7µm nylon screens. Dissociated islets were stained with antibodies against HLA-DR-Bv421 (L243, BioLegend) and CD45-FITC (HI30, BioLegend) in FACS buffer with 2% serum. PI-/CD45-/HLA-DR+ or PI-/CD45+/HLA-DR+ Mice Pancreata were perfused with 0.5mg/mL of collagenase P (Sigma-Aldrich) through the common bile duct and digested at 37ºC for 17min. After several washes, pancreatic slurry was passed through a 0.419mm wire mesh strainer. A density gradient was established by layering histopaque1077 (Sigma-Aldrich) and serum-free RPMI, and sample loaded on top. After centrifugation at 600 g for 20 min, islets were collected at the histopaque/media interface, washed and hand-picked under a dissecting microscope. An average of 300-400 islets were recovered per mouse. For making single cell suspension, islets were digested with trypsin-EDTA and washed extensively before being counted.Dissociated islets were stained with antibodies against MHC II-PE (OX-6, BioLegend) and CD45-FITC in FACS buffer with 2% FBS. PI-/CD45-/MHC II+ or PI-/CD45+/MHC II+ cells were sorted into semi-skirted 96 well PCR plates with 5ul of RT solution per well.