Ly6c percp cy5

To measure the dynamic changes in the proportions of immune cell populations in the peripheral blood of rats, 50 μl of blood samples was taken for flow cytometry analysis. After lysing red blood cells, the remaining leukocytes were resuspended in PBS containing 2% FBS, and then were stained with HIS48-FITC (eBioscience), CD11bc-allophycocyanin (BioLegend), CD68-PE (Biolegend) or CD206-PerCP (Abcam) for 30 min at 4°C. On the other hand, mice cells were analyzed by staining with CD11b-FITC (BD Bioscience), Gr-1-PE, Ly6G-APC (BD Bioscience), Ly6C-PerCP-Cy5.5 (eBioscience), F4/80-PE (eBioscience) or CD206-PerCP (Biolegend, #141716). Acquisition of flow cytometry data from the BD FACSCantoII flow cytometer was performed using FACSDiva software (BD Biosciences). The number of events analyzed was 10,000 per sample. Analysis was performed using FlowJo software (Tree Star).

Lung tissue was chopped and digested with collagenase-D (1 mg/ml; Roche) and DNase I (10 mg/ml; Sigma-Aldrich) for 1 h at 37°C with agitation. To detect cytokines, brefeldin A (5 µg/ml) was added during the digest. Lungs or spleens were passed through a 70-µm cell strainer to a obtain single-cell suspension, followed by RBC lysis with ACK buffer. The cells were incubated with Fcγblock (anti-CD16/CD32 antibody, BD Biosciences) (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. The following surface Abs were used: CD69-FITC, CD3-APC-ef780, MHCII-APC, Ly6C-PerCP-Cy5.5, CD86-FITC, CD11b-APC-ef780, CD38-ef450, F4/80-PE-Cy5, CD49d-PerCP-ef710, CD3-AF700, CD8-APC-ef780, CD44-PE-Cy7, CD4-PE-Cy5 (eBiosciences), CD44-BV605, CD4-BV785, CD103-PE, Ly6G-BV605, CD80-PE-Dazzle594 (BioLegend), CD62L-PE-CF594, SiglecF-PE, CD103-BV786 (BD Biosciences). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–V450 (BD Biosciences). Fluorescence minus one samples were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).

For flow cytometric analysis, cells were fixed in fixation/permeabilization buffer (eBioscience) overnight, washed and blocked in PBS containing 1% bovine serum albumin (BSA) and rat immunoglobulin (1µg/ml, Sigma, St. Louis, USA). Cells were stained with F4/80 APC or PerCP-Cy5.5, CD11b APC or FITC, Gr1 PE-Cy7, CD80 FITC, CD86 PE or APC, MHC-II PE or FITC, CD40 PE, Ly6C PerCP-Cy5.5 (all eBioscience) and SiglecF PE (BD Biosciences). To stain for RELMα-positivity, cells were pre-incubated in permeabilization buffer (eBioscience) and then stained with anti-RELMα (Peprotech, New Jersey, USA). Subsequently, cells were washed twice in permeabilization buffer and a secondary antibody (goat anti-rabbit Alexa488, Invitrogen, Carlsbad, USA) was used. As a control unspecific and isotope identical Alexa488 antibody (Invitrogen) was used. Data was acquired using a BD FACS Canto and BD FACSDiva software; for generation of figures and plots FlowJo software (Tree Star, Ashland, USA) was used.

To obtain individual splenic cells, murine spleens were mechanically dissociated, passed through a 75‐μm cellular sieve and then through a 30‐μm nylon mesh (Catalog number: 130‐041‐407; Pre‐Separation Filters; MiltenyiBiotec, Bergisch Gladbach, Germany). Red blood cells (RBC) were lysed with ACK Lysis Buffer (Catalog number: 420301; BioLegend, San Diego, California, USA). Cells were washed with phosphate buffered saline (PBS) and counted. Splenic cells were marked with specific fluorophore‐conjugated anti‐mouse antibodies in PBS for 15 minutes in the dark. Fluorophore‐conjugated antibodies used in this study include CD11b‐APC (Catalog number: 17‐0112‐81; M1/70; eBioscience, San Diego, California, USA), Ly6G‐PE (Catalog number: 551461; 1A8; BD Biosciences, Franklin Lakes, New Jersey, USA), and Ly6C‐PerCP‐Cy5.5 (Catalog number: 45‐5932‐82; HK1.4; eBioscience). M‐MDSCs were isolated by MoFloFlow Cytometry (Dakocytomation), and cell debris was excluded using a SSC/FSC gate. CD11b‐positive cells were gated, and then Ly6G⁻Ly6C^high cells were isolated as M‐MDSCs or their counterparts (from tumor‐free mice) and Ly6G⁺Ly6c^+/low cells as G‐MDSCs or their counterparts. Purity was over 90%.