Anti cd8a

Single-cell suspensions were resuspended in BD stain buffer (BD Bioscience) for 15 min prior to staining with specific antibodies. Antibodies against cell markers: anti-CD45, anti-CD3, anti-CD4, anti-CD8a, anti-CD11b, anti-F4/80, anti-Ly6G, anti-Ly6C, anti-NKp46(CD335), anti-B220, and anti-CD69, were purchased from BD Bioscience and BioLegend; anti-OX40L and anti-41BBL from Miltenyi Biotec (detailed in Supplementary table 4). Samples were mixed with FVS700 (1/7000) and data were acquired on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star). ROS generation in cultured neutrophils was determined using either DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851) following the manufacturer’s protocol or a dihydroethidium (DHE) fluorescent probe. Cells were incubated with DHE (10 μM) in HBSS containing 1.5 mM CaCl₂ and 1 mM MgCl₂ for 30 min at 37 °C and analyzed by flow cytometry. LacZ activity was determined by flow cytometry using Fluorescein di[β-D-galactopyranoside (Sigma, F2756) as described elsewhere^{94 (link)}.

The protocol of above mentioned blood sampling was as follows. All antibodies were purchased from BioLegend. Antibodies were directly marked with one of the following fluorescent tags: fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC). anti-CD3(Cat: 552774, BD bioscience), anti-CD(Cat: 550954, BD bioscience), anti-CD8a(Cat: 557654, BD bioscience) antibodies and isotypes were used to react with rat antigens. Cell surface phenotype and sorting were recognized using a flow cytometer (BD company, USA), and the data were analyzed using FlowJo 7.6.1 software.

Mouse pancreatic tissues were digested by digestion buffer (1 mg/ml collagenase IV [Sigma-Aldrich] and 10 U/ml DNase I [Sigma-Aldrich] in DMEM) at 37 °C for 30 min. The digested tissues were pushed through a 70-µm cell strainer and washed by 40 ml of DMEM without FBS. The digested cells were resuspended in 37% Percoll, with the same volume of 70% Percoll on the bottom of the tube. After centrifugation at 800 g for 20 min with break off, the immune cells were isolated from the 37%/70% interface and rinsed by PBS. The cells were first stained by Zombie UV (BioLegend, #423107, 1:500), then were incubated with a cocktail comprising the following antibodies in cell staining buffer (#420201, BioLegend): anti-CD45 (#103131, 1:150), anti-CD3ε (#100355, 1:60), anti-B220 (#103231, 1:200), anti-Gr1 (#108438, 1:150), anti-CD11b (#101215, 1:250), anti-F4/80 (#123131, 1:400), anti-Ly6C (#128033, 1:40), anti-Ly6G (#127651, 1:80), anti-CD4 (#100405, 1:200), anti-IA/IE (#107607, 1:500, all from BioLegend), and anti-CD8a (#564297, 1:200, BD Biosciences). After washing, the stained cells were submitted for multiple-color flow cytometry analysis. The data were collected on LSR Fortessa X-20 analyzer with FACSDiva software version 8.0 (BD) and analyzed using FlowJo version 10 (BD).