Rat anti mouse cd11c pe cy7

BMDCs treated with SyBV for 24 h were blocked for non‐specific staining with 2.4G2 (anti‐Fc‐receptor) for 30 min at 4°C in PBS/0.1% BSA and then washed and stained for 30 min at 4°C in PBS/0.1% BSA with the following antibodies: rat anti‐mouse MHCII‐ BV421 obtained from eBioscience; rat anti‐mouse CD11c‐PE‐Cy7, rat anti‐mouse CD83‐PE (Michel‐19), and rat anti‐mouse CD40‐APC‐Cy7 obtained from BD Biosciences; and rat anti‐mouse CD86‐BV510 obtained from BioLegend. To exclude dead cells, 7‐aminoactinomycin D (Sigma Aldrich)‐stained cells were excluded from the analysis. Events were collected and analysed using a Fortessa‐X20 Flow cytometer (BD Biosciences) and FlowJo Software (Tree Star Inc.). For signalling molecule analysis, BMDCs were treated with SyBV plus tEV for 5–30 min. Whole‐cell lysates were separated by 10% SDS‐PAGE and transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti‐beta‐actin antibody (Sigma Aldrich) or anti‐phospho‐p65 (serine 365; Cell Signaling Technology). After incubation with horseradish peroxidase‐conjugated secondary antibody, the immunoreactive bands were visualized with a chemiluminescent substrate.

Mouse lung was digested and single-cell suspensions were prepared as previously described (46 (link)). Cell suspensions underwent red blood cell lysis using Pharm Lyse buffer (BD Biosciences). Live/dead staining was performed in protein-free solution (HBSS) using fixable viability dye eFluor 506 (eBioscience), followed by incubation with FcR-blocking reagent (Miltenyi Biotec). Antibodies utilized for murine cell staining included rat anti–mouse CD45–FITC (BioLegend, 30-F11), rat anti–mouse Ly6C–eFluor450 (eBiosciences, HK1.4), rat anti–mouse I-A/I-E–PerCPCy5.5 (BioLegend, M5/114.15.2), rat anti–mouse CD45–APC (BioLegend, 30-F11), rat anti–mouse Ly6G–Alexa Fluor 700 (BioLegend, 1A8), rat anti–mouse NK1.1–Alexa Fluor 700 (BD, PK136), rat anti–mouse CD11b–APCCy7 (BioLegend, M1/70), rat anti–mouse CD64–PE (BioLegend, X54-5/7.1), rat anti–mouse SiglecF–PECF594 (BD, E50-2440), and rat anti–mouse CD11c–PECy7 (BD, HL3). For neutrophil quantification, 123Count eBeads (Invitrogen) were added. Flow analysis of fixed samples was done on a BD FACSymphony A5-Laser Analyzer at the Northwestern University Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core facility. Acquired data were analyzed with FlowJo v10.8 (FlowJo).