Bp0075 1

Rat anti–mouse Ly6G antibody (BP0075-1, BioXcell) was used for neutrophil depletion (100 μg/mice, i.p.) as described (23 (link)). 5×10⁵ MC-38 cells were injected into the caudal vein of each BALB/c mouse, in the presence or absence of PAD2-IN-1 (20 mg/kg, i.p.). The presence of METs was detected by IF staining, as above described. The weights of livers were measured to assess the actual tumor burden. The number of nodules on the livers was counted and confirmed by HE staining. All animal experiments were approved by the Medical Ethics Committee of Shandong University.

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.

Lungs were harvested from mice infected with Mabc for the indicated times, fixed in 10% formalin, and embedded in paraffin wax. For histopathology, lung paraffin sections (4 µm) were cut and stained with hematoxylin and eosin (H&E). H&E-stained images were scanned using the Aperio Digital Pathology Slide Scanner (Leica, Wetzlar, Germany) and imaged using the ScanScope® CS System (Leica). The histopathological severity score was graded by scanning multiple random fields in six sections per mouse. For immunohistochemical (IHC) staining, lung paraffin sections (4 µm) were cut and immunostained with antibodies specific for myeloperoxidase (MPO; Abcam, Cambridge, MA, USA, ab9535) and LyG6 (Bio X Cell, West Lebanon, NH, USA, BP0075-1). Immunohistochemically stained lung tissue slides were examined using a confocal laser scanning microscope.

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.

This antibody was used to deplete neutrophils in vivo as previously described (29 (link)). Mice were injected intraperitoneally with 500 μg/injection of antibody every 3 days over a time course starting from 2 days before the start of doxorubicin administration until the end of the study as described previously (29 (link)). Isotype control anti-Trinitrophenol antibody (rat IgG2a) was similarly injected into a control group of mice. Antibody against Ly6G (clone 1A8) and rat IgG2 control antibody (clone 2A3) were purchased from BioXcell (catalog BP0075-1 and BP0089, respectively). These antibodies were stored at 4°C, and the solutions were mixed with PBS before injection (total volume of 200 μL). Injections were performed using a sterile syringe and 29-gauge needle. The expiration date of the mixtures coincided with the expiration date of the antibodies.

For granulocyte depletion, mice were injected once intraperitoneally with 0.1 mg of IgG control (clone 2A3) or anti-Ly6G antibody (BP0075-1; BioXCell) in 200 µl PBS. For short-term in vivo lineage tracing assays, CD45.1 recipient mice were sublethally irradiated (8.5 Gy, delivered in split doses 3 h apart) using an x-ray irradiator (MultiRad225, Precision X-Ray Irradiation) and injected retroorbitally with 5,000 CD45.2 donor cells within the next 6 h. Irradiated recipient mice were administered polymyxin/neomycin-containing water for 4 wk following transplantation to prevent opportunistic infection and analyzed over time by repeated bleedings. Peripheral blood (PB) was obtained from retro-orbital plexus and collected in tubes containing 4 ml of 10 mM EDTA in ACK (150 mM NH₄Cl/10 mM KHCO₃) lysis buffer for flow cytometry analyses. For short-term in vivo differentiation assays, 10,000 donor cells isolated from β-actin-Gfp mice were retro-orbitally infused into recipient mice, which were analyzed for BM contribution at the indicated times.