Pe conjugated f4 80

To analyze M1Mϕ and M2Mϕ polarization, BMDMs were probed with antibodies against phycoerythrin (PE)-conjugated F4/80 (565410; BD Pharmingen, San Diego, CA, USA), FITC-conjugated CD11b (557396; BD Pharmingen), PE-Cy7-conjugated CD11c (558079; BD Pharmingen), and Alexa Fluor-647-conjugated CD206 (565250; BD Pharmingen) at room temperature. TECs were stained using the propidium iodide necrosis detection kit (eBioscience, San Diego, CA, USA). All stained cells were analyzed by flow cytometry (BD Biosciences) using FlowJo software (v.10.0; Tree Star, Ashland, OR, USA).

150 μL peripheral blood and the hind ankles were collected on day 5 and 13 post KBx/N serum transfer, respectively. For the peripheral blood samples, red blood cells were lysed and cells were prepared as single cell solution. The hind ankles were cut into 1 mm² pieces, and then digested in 10% DMEM containing Collagen IV (1 mg/ml) for 1 h at 37°C. Single cell solution was then prepared after passing through 40 μm cell strainer. 1×10⁶ cells were stained with FITC-conjugated CD11b (BD, United States, 1:500), PE-conjugated F4/80 (BD, United States, 1:400), PerCP-Cy5.5-conjugated Ly6G (BD, United States, 1:400) and APC-conjugated Ly6C (BD, United States, 1:200) to detect macrophage, neutrophil and monocyte. Data were acquired using a BD FACSCalibur flow cytometer (BD Biosciences) and were analyzed by using FlowJo software (Version 7.6.1).

After being blocked with CD16/CD32 (Cat. 553,141, BD Pharmingen™, USA), single-cell suspension (1 × 10⁶ cells) was incubated with a flow panel for 30 min avoiding light, the panel included APC-CY7 conjugated CD45 (Cat. 557,659, BD PharmingenTM, USA), PE-conjugated F4/80 (Cat. 565,410, BD PharmingenTM, USA), APC-R700 conjugated CD86 (Cat. 565,479, BD PharmingenTM, USA), and AF-647 conjugated CD206 (Cat. 565,250, BD PharmingenTM, USA). The ratio of Panc02 cells to M0 macrophages was then calculated using flow cytometry (CytoFLEXS, Beckman COULTER, USA) to identify the single-cell suspension.