Facs analyzer lsr

Islets were isolated by collagenase digestion, hand-picked, and incubated overnight at 37 °C. After stimulation with or without IL18 (100 ng/mL, B004-2, MBL International) and 50 μM adenosine dialdehyde (Adox, A7154, Sigma-Aldrich) for 72 hrs as previous reported (Bramswig et al., 2013 (link)), islets were dispersed with accutase (A1110501, GIBCO, Gaithersburg, MD) for 10 min at 37 °C, and resuspended in FACS buffer. After 15 min incubation with an Fc block (16-0161-82; eBioscience), islet cells were stained with Alexa Fluor 647 anti-insulin (565689, BD Pharmingen, Woburn, MA) and PE anti-glucagon (565860, BD Pharmingen) or isotypes for 30 min at 4 °C with intracellular staining buffer according to the manufacturer’s instructions (00-5523, Thermo Fisher Scientific). Islet cells were initially selected by size on the basis of forward scatter (FSC) and side scatter (SSC), and then separated on the basis of cell-surface markers using a FACS Analyzer LSR (BD Biosciences).

Cells from peritoneal cavity and blood were resuspended in PBS with 2% FBS after red blood cell lysis and washed with PBS, and incubated with FACS antibodies for 30 min on ice. Cells were initially selected by size on the basis of forward scatter (FSC) and side scatter (SSC), following separated on the basis of cell-surface markers using a FACS Analyzer LSR (BD Biosciences). To detect macrophage percentage in peritoneal cavity and blood, we stained macrophages with anti-CD45-FITC (1:100, #11–0451-82, eBiosciences), anti-CD11b-APC and anti- F4/80-PerCP-Cyanine5.5. To detect monocyte, we stained cells with anti-CD45-PerCP-Cyanine5.5 (1:100, #45–0451-82, Invitrogen), anti-CD11b-APC, and anti- Ly6C-FITC (1:250, #53–5932-82, Invitrogen). To detect mast cells, we stained cells with anti-CD45-PerCP-Cyanine5.5, anti-c-kit-APC (1:100, #17–1171-82, eBiosciences) and anti-FcεR1-FITC (1:250, #11–5898-82, eBiosciences). To detect basophils, we stained cells with anti-CD45-PerCP-Cyanine5.5, anti-CD63-PE/Cy7 (1:100, #143910, BioLegend), and CD200R3-PE (1:100, #142206, BioLegend).