Af488 conjugated goat anti rabbit

For surface staining, cells were stained with antibody to the following markers: CD45.2, CD4, CD8, CD11b, F4/80, CD11c, Ly6C, and Ly6G (BioLegend, San Diego, CA). For intracellular STING staining, after surface staining, cells were fixed and permeabilized with a kit (eBioscience, San Diego, CA) according to the manufacturer’s guidelines. Rabbit unconjugated STING antibody and Rabbit immunoglobulin G isotype control (Invitrogen, Carlsbad, CA) were used as primary antibody, and AF488 conjugated goat anti-rabbit used as secondary antibody (Life Technologies, Carlsbad, CA). Dead cells were excluded from analysis with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA). Cells were run on LSRII (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Treestar Inc, Ashland, OR).

Isolation of pancreatic leukocytes was through collagenase digestion of mouse pancreas as previously described^{13 (link)}. Dead cells were stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermofisher scientific, Santa Clara, CA). For surface staining, cells were stained with antibody to the following markers: CD45, CD4, CD8, CD11b, F4/80, CD11c, CD44 and CD45RB (BioLegend, San Diego, CA). For intracellular staining, cells were stained with IFNγ, IL-4 and foxp3 (Biolegend, San Diego, CA), IL-17A and RORγt (eBioscience, San Diego, CA). Intracellular STING staining was as previously described¹⁰ , cells were stained with surface markers first, then fixed and permeabilized with a kit reagents from eBioscience (San Diego, CA). Then Rabbit unconjugated STING antibody or Rabbit IgG isotype control (Invitrogen, Carlsbad, CA) were used as primary antibody and AF488 conjugated goat anti-rabbit was used as secondary antibody (Life Technologies, Carlsbad, CA).