Bv421 tgfβ

Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat #12430-054) and Penicillin/Streptomycin (Cat #15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) and G418 sulfate were obtained from Invitrogen (Carlsbad, CA). Rabbit antibody against β-actin (Cat #4970S) was from Cell Signaling (Beverly, MA). Rabbit antibody against NHE1 (Cat #ab67314) and rat antibody against CD8 (Cat #ab22378) were from Abcam Ltd. (Cambridge, MA). Rat anti-mouse CD31 (Cat #550274) was from BD Pharmingen (San Jose, CA). Rabbit anti-ionized calcium-binding adapter molecule 1 (iba1) was from Wako (Richmond, VA). PerCP/Cy5.5-CD45, PE-P2RY12, BV421-TGFβ, BV605-TNFα, APC/780-IL-1β, PE/Cy7-IL10, PerCP/Cy5.5-CD8a, APC/Cy7-CD4, Alexa Fluor 700-CD25, PE-FoxP3, BV605-PD-1, PE-Gr-1, APC-NK1.1, and Pacific Blue Granzyme B were obtained from Biolegend (San Diego, CA). eFluor 450-CD16/32, PE/Cy7-CD206, APC-IFNγ was purchased from eBioscience (San Diego, CA). APC-IL-6 were obtained from thermos fisher (Waltham, MA). BUV737-CD11b, Alexa Flour 700-CD86 were obtained from BD Biosciences (San Jose, CA). PE-Ym1were from Abcam Ltd. (Cambridge, MA). Rabbit anti-Laminin (Cat #L9393) and cariporide (HOE642) was purchased from Sigma Chemicals (St. Louis, MO). Anti-mouse PD-1 (RMP1-14) and IgG2a isotype control were from BioXcell (West Lebanon, NH).

Antibodies specific for mouse PerCP-cy5.5-CD45, FITC-CD11b, PE-Ly6C, BV510 or BV421-Ly6G, BV421-TGFβ, anti-IL-10 and anti-VEGF were purchased from Biolegend. Anti-human APC-CXCR2 antibodies and anti-rat FITC-secondary antibodies were purchased from BD Pharmingen. Anti-mouse CXCR2 antibodies were obtained from R&D Systems and LIVE/DEAD Fixable Blue Dead Cell Stain was obtained from Thermo Fisher Scientific.
To analyse the neutrophils and their functions in tumor tissues, tumors were minced into small pieces and dissociated using 1 mg/mL collagenase type IV (Sigma-Aldrich) in serum-free DMEM medium at 37°C for 1 h. After washing with PBS, cell suspensions were filtered with a 70-μm nylon cell strainer (BD Falcon) to remove clumps of cells and debris for subsequent flow cytometry. For cell surface staining, cells were stained with antibodies on ice for 30 min in the dark; for intracellular cytokine staining, cells were then fixed and permeabilized with paraformaldehyde and Triton-X100 and stained with intracellular antibodies overnight. Analyses were carried out on a NovoCyte flow cytometer (ACEA Biosciences) and data were analyzed using Novo Express 1.1.2.