Bp0089

Anti-mouse Ly6G (300 μg; clone IA8; BP0075-1; BioXCell) or its isotype control rat IgG2a (clone 2A3; BP0089; BioXCell) in 1× PBS was administered via the i.p. route to LPS/saline mice. Antibody injections began the same day as LPS/saline injections and continued every 48 h. CFU numbers were assessed at 7 days of infection. Single-cell suspensions were isolated at day 0 (uninfected) and analyzed by flow cytometry to assess depletion efficiency. Intracellular Ly6G was used in place of surface Ly6G to account for potential surface antigen masking by the depletion antibody (70 (link)). After 2% PFA fixation, intracellular Ly6G was stained in the intracellular staining permeabilization wash buffer (BioLegend) using the manufacturer’s protocol. Ly6G antibodies (Ly6G, PE; clone 1A8; BD Pharmingen) used for intracellular staining were diluted half of what was typically used for surface staining.

4T1 or E0771 cells were implanted as described above. On day 7 after implantation, mice were started on the OLT1177 diet or continued on standard diet. At day 10, a neutralizing antibody against PD-1 (RMP1.14) (Catalog#BP0146) (8 mg/kg in 200 μL saline; BioXCell, Lebanon, NH, USA) or IgG (8 mg/kg in 200 μL saline; BioXCell) (Catalog#BP0089) was injected. Mice were sacrificed 21 days post tumor implantation.

Four-month-old 129S6/SvEvTac Pkd1^RC/RC mice were treated twice a week for 8 weeks by i.p. injection with 10 mg/kg anti–PD-1 blocking antibody (clone RMP1-14; Bio X Cell, BP0146) or 10 mg/kg IgG2a control (clone 2A3; Bio X Cell, BP0089). C57BL/6J Pkd1^RC/– mice were treated every other day by i.p. injection with 10 mg/kg anti–PD-1 blocking antibody or 10 mg/kg IgG2a control starting at P8 until P20.

Wild-type (WT) C57BL/6, CXCR2−/−, HIF-1αfl/fl and MRP8cre+ mice were obtained from The Jackson Laboratory, bred and maintained under specific pathogen-free (SPF) conditions in the Biological Resource Centre (BRC) of A*STAR, Singapore. HIF-1αfl/fl mice (The Jackson Laboratory, Bar Harbor, ME, USA) were a kind gift from Dr. Subhra K. Biswas at the Singapore Immunology Network. Male and female mice were used, and all experimental groups were gender-matched and age-matched. HIF-1αfl/fl and MRP8cre+ mice were crossed in-house to generate progeny with HIF-1α-deficient neutrophils (HIF-1αΔNφ). All experiments were performed under the approval of the Institutional Animal Care and Use Committee (IACUC, Singapore) of the BRC, in accordance with the guidelines of the Agri-Food and Veterinary Authority (AVA, Singapore) and the National Advisory Committee for Laboratory Animal Research (NACLAR, Singapore) of Singapore (IACUC protocol #171230). In some experiments, Ly-6G (Bio X Cell; Clone 1A8, BP0075) or isotype control antibody (Bio X Cell; BP0089) was administered via intraperitoneal (i.p.) injection at 200 ug per 20 g mouse three times per week.

For subcutaneous tumors, 2.5 × 10⁵ cells, unless indicated otherwise, were injected into the right flank of mice. Tumor volume was measured with a caliper every 2 or 3 d and calculated according to the formula V = (L × W²)/2. Mice that were developing a tumor-associated skin ulcer during the experiment were humanely killed, and visually ulcerated tumors were not included in further analysis. For the hepatic metastasis model, 2.5 × 10⁴ to 1.0 × 10⁵ luciferase-expressing cells were injected into the portal vein. Tumor size was monitored through bioluminescence imaging every 3 or 4 d. To do so, 150 μL of 30 mg/mL D-Luciferin (PerkinElmer, 122799-10) was administered through intraperitoneal injection, and mice were imaged 10 min after D-Luciferin administration with the IVIS Spectrum In Vivo Imaging System. For mouse tumor immunotherapy, 200 μg of isotype IgG (BioXcell, BP0089) or rat anti–mouse PD-1 antibody (BioXcell, BP0273) was given through intraperitoneal injection on the indicated days. All mice that were developing tumor-associated ascites or lethargy during the experiment were humanely killed. For the ovalbumin-inducible 1242-sgScramble and 1242-sgKRT19 s/c tumors, 5 × 10⁵ cells were inoculated, and ovalbumin was induced using Dox food at day 0.

The animals were lightly anesthetized with isoflurane (Pivetal®) and subcutaneously implanted with 25-mg slow-release morphine pellet or placebo pellet. The pellets were obtained from National Institute on Drug Abuse. All efforts were made to minimize suffering during and after surgery. To deplete gut microbiota, a pan-antibiotics + antifungal cocktail [vancomycin 32 (mg/kg), bacitracin (80 mg/kg), metronidazole (80 mg/kg), neomycin (320 mg/kg), Pimaricin (0.192 mg/kg)] was prepared every day in drinking water and an oral gavage was given to mice for 8 days prior to treatment with morphine or placebo pellet as described previously.^{27 (link)} For neutrophil depletion assays, 150 μg of anti-Ly6G (clone 1A8, # BP0075-1, Bio X Cell) and corresponding isotype control (clone 2A3, # BP0089, Bio X Cell) were injected intraperitoneally 3 days prior to morphine or placebo pellet implant with dose as described in the schematic in figure or legends.