Rat igg2a isotype control clone 2a3

All B16-derived cell lines were washed twice in PBS, filtered using a 70 μM nylon filter to remove cell aggregates. To evaluate subcutaneous tumor growth, 1 × 10⁵ cells were inoculated subcutaneously in the flank. Tumor growth was monitored every 2–3 d until the maximal diameter of 2 cm. Tumor volume was calculated using the modified ellipsoidal formula: ½(length × width²). To evaluate tumor formation in the lung, each mouse received 2 × 10⁵ cells in 100 μL PBS via the tail vein. All injected mice were euthanized 17–20 d later. Lung tissues were excised, photographed and the tumor nodules counted. To deplete CD8⁺ T cells, anti-CD8⁺ (clone 53-6.72) or isotype control (Rat IgG2a, clone 2A3) monoclonal antibodies purchased from BioXCell (West Lebanon, NH, USA) was administered via intra-peritoneal route (25 μg/mouse). Depletion of CD8⁺ T cells was confirmed 24 h later by flow cytometry. Antibody-treated mice were inoculated with B16-5 cells subcutaneously or intravenously. For immunization, B16 cells were irradiated, washed, suspended in PBS and 2 × 10⁵ cells in 40 μL volume were injected via intradermal route on the left flank of anesthetized, 6–8-weeks-old C57BL/6 mice. Immunized mice were challenged 4–5 weeks later with 1 × 10⁵ parental B16 cells, inoculated subcutaneously on the right flank or 2 × 10⁵ cells intravenously. Tumor growth was monitored as described above.

To establish the adenocarcinoma tumor models C57BL/6 WT female mice at 8–10 weeks of age, were intravenously injected with 7 × 10⁴ KP cells or with 2 × 10⁵ KP OVA variants (WT or KO^CXCL5, KO^{CXCL5 (lenti-CXCL5)} or KO^{CXCL5 (lenti-vec)}). In one experiment (supplementary Figure 5) mice were challenged with 5 × 10⁴ cells. Otherwise indicated, mice were sacrificed at initial (9 days) or at established stage of tumorigenesis (18 d). To deplete neutrophils in vivo, KP OVA bearing mice were intraperitoneally treated every 3 d, starting from the 6th to 12th d, with 200 µg of anti-Ly6G antibody (InVivo Plus, clone 1A8, Bio X Cell) or isotype control (Rat IgG2a isotype control, clone 2A3, Bio X Cell) and sacrificed at d 13.
In PD-L1 blockade experiments, mice were challenged with KP OVA WT or KO^CXCL5 cells, intraperitoneally treated every 3 d with 200 µg of αPD-L1 (InVivoMab, clone 10 F.9G2, BioXcell) or isotype control (InVivoMab, rat IgG2b isotype control, clone LTF-2, BioXcell). Mice were sacrificed at d 18.

Four-week-old CBA/J mice were treated with 200 µg rat anti-mouse IL-17A monoclonal antibody (MAb) (clone M210; Amgen) or rat IgG2a isotype control (clone 2A3; BioXcell) at 3 days before and on the day of BMDC transfer (500,000 intraperitoneally from mice receiving SFB gavage or those not receiving SFB gavage). Mice were challenged intracecally with 2 × 10⁶ trophozoites 7 days after BMDC transfer.

Exponentially growing C51 and CT26 mouse colon carcinoma cell lines (kindly provided by Philogen S.p.A.) syngeneic to the Balb/c mice were cultured in Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) (Lonza), respectively, supplemented with 10% fetal calf serum (FCS) in a humidified 5% CO₂ chamber at 37°C.
The L19-IL2 immunocytokine (Philogen S.p.A.) was diluted with sterile phosphate buffered saline (PBS, Lonza) to concentrations of 200 µg/ml. For in vivo depletion experiments, the monoclonal antibodies anti-CD8a (clone YTS 169.4), anti-CD4 (clone YTS 191) and the isotype control anti-KLH rat IgG2b (clone LTF-2) (BioXCell) were diluted with sterile PBS to a concentration of 1.67 mg/ml. For in vivo blocking experiments, the monoclonal anti-CD127 (clone A7R34) and rat IgG2a isotype control (clone 2A3) antibodies (BioXCell) were diluted with sterile PBS to a concentration of 2 mg/ml. All studies were conducted in a laboratory that operates under exploratory research principles using established laboratory protocols and general research investigative assays.