Celesta analyzer

Pancreata were minced and digested in 5 ml DMEM/F12 (without phenol red and supplemented with 5 mg/ml d-glucose) in the presence of collagenase IV (1 mg/ml, StemCell Technologies #07909), dispase (1 U/ml, StemCell Technologies #07923), hyaluronidase (100 U/ml, Worthington Biochemical), and DNase type I (100 U/ml, Sigma-Aldrich) for 2–3 h at 37 °C with periodic mixing. Dissociated cells were washed twice in PBS prior to filtering out debris using a 70 μm mesh filter. Single cells were resuspended in flow staining buffer (5% horse serum in PBS) and stained with fluorochrome-conjugated antibodies against T-cell marker CD3e (BV-421; clone 145-2C11), B cell marker B220 (APC; clone RA3-6B2), dendritic cell marker CD11c (PE; clone N418), and macrophage cell marker CD11b (FITC; clone M1/70). Cell viability was assessed with SYTOX™ Blue Dead Cell Stain (ThermoFisher #S34857). Data collection took place on a 3-laser BD Celesta analyzer and analysis was performed with FlowJo software (TreeStar). Supplementary Table 2 provides the catalog numbers and dilutions of antibodies.

For analysis of apoptosis by Annexin V, PC-9 cell lines were washed with HBSS and detached by trypsinization. DMEM was added to the cells and the cells were incubated at 37 °C for 30 min with occasional shaking to allow the cell membranes to recover from the trypsin treatment while keeping the cells detached. Following collection, a single cell suspension of 1 × 10⁶ cells/ml was prepared in Annexin V Binding Buffer (422201, Biolegend, San Diego, CA). While protected from light, samples were incubated first with FITC-Annexin V (640906, Biolegend) for 15 min at room temperature and then with Sytox Red dead cell stain (50-112-1578, Fisher Scientific) for an additional 15 min at room temperature. The samples were subsequently analyzed by flow cytometry using the 3-laser BD Celesta Analyzer. Analysis was performed by FCS Express.

For surface marker staining, mononuclear cells were previously blocked with anti-CD16/32 antibody (Thermo Fisher Scientific) for 15 min and then incubated with the following anti-mouse antibodies: PE-CD19, PE-B220 and Pacific blue-CD45 (all purchased from Thermo Fisher Scientific).
For intracellular marker staining, cells were previously stimulated with 50 ng/mL PMA and 750 ng/mL ionomycin in the presence of 20 µg/mL brefeldin A for 5 h, then surface-stained for 30 min. After surface marker staining, cells were resuspended in Fixation/Permeabilization Buffer Set (Thermo Fisher Scientific) and stained with PE-Cy7-IL-10 (BioLegend, San Diego, CA, USA); APC-IL-12A (Thermo Fisher Scientific) and FITC-EBI3 (Novus Biologicals, Littleton, CO, USA) for 45 min according to the manufacturer’s protocol.
For intranuclear (p-STAT3) marker staining, cells were previously surface stained and then fixed and permeabilized using a transcription factor staining buffer set (Thermo Fisher Scientific) and stained with PE-p-STAT3 (Thermo Fisher Scientific) for 45 min according to the manufacturer’s protocol.
Isotype antibodies were used as negative controls. Flow cytometry analysis was performed using BD Celesta Analyzer.