Recombinant mouse m csf

Male C57BL6/J mice (10-weeks-old) were used to prepare peritoneal macrophages and bone marrow derived macrophages (BMDMs). The peritoneal macrophages were obtained by flushing the peritoneal cavity two times with 5 mL of ice-cold culture medium containing RPMI-1640 (Nacalai Tesque, Kyoto, Japan), 10% FBS, 0.1% 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin-streptomycin mixed solution (Nacalai Tesque) using a 25-gauge needle. The peritoneal fluid collected was poured into a 100 mm dish, followed by incubation for 4 hr in a CO₂ incubator to isolate the cells attached to the bottom of the dish. After incubation, the dish was washed two times with 10 mL of pre-warmed culture medium, with the attached cells then collected by gentle scraping into 10 mL of culture medium for use in the experiments as peritoneal macrophages. For preparation of BMDMs, bone marrow cells were collected from femurs and tibias by flushing these bones with 10 mL of culture medium. After lysis of the red blood cells with 2 mL of hypotonic ammonium chloride solution, the cells were collected by centrifugation at 500 x g for 5 min at 4°C, and then cultivated for 7 d in 10 mL of culture medium containing recombinant mouse M-CSF (Pepro Tech, Cranbury, NJ, USA) at a concentration of 10 ng/mL.

Macrophages was generated as previously described [27 (link)]. Briefly, bone marrow cells were isolated from the femurs and tibias of C57BL/6 mice and then seeded in cell culture dishes and cultured with complete DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 20 ng/mL recombinant mouse M-CSF (PeproTech) at 37°C in a CO₂ incubator for 5 days to differentiate into macrophages. The cells were washed twice with phosphate-buffered saline (PBS) every other day, and fresh medium was added. On day 6, nonadherent cells were removed and adherent cells were cultured with 20 ng/mL mouse recombinant IL-4 (PeproTech) or 1 μg/mL LPS (Sigma) for 24 h, either with or without L-4F (0.25 μg/mL). The differentiated, adherent, live macrophage population was detached from the plate with a solution containing trypsin (Gibco), and the cells were processed for phenotypic characterization. Based on the specific expression of a number of surface markers, including CD11b, F4/80, MHC II and CD206, the cells were sorted using flow cytometry.

Bone marrow (BM) cells were obtained from femurs and tibias of 8–10-week-old mice and differentiated in RPMI medium supplemented with 10% FCS and recombinant mouse M-CSF (PeproTech 315-02, lot 0914245) at 100 ng/mL for up to 7 days. At the end of the culture period, the cells were differentiated in BMDMs; 100% of adherent cells were positive for F4/80 antigen as measured by flow cytometry. Hmox1 tissue-specific deficient BMDMs were treated with 4-hydroxytamoxifen (Sigma H7904) 24 h before starting the experiments.

To generate bone marrow-derived macrophages (BMDMs), bone marrow cells were isolated from the femurs of C57BL/6 mice and red blood cells were lysed with ammonium chloride potassium lysis buffer. Cells at a concentration of 1 × 10⁶ cells/ml were cultured in CM supplemented with 20 ng/ml recombinant mouse M-CSF (PeproTech, USA) for 7 days, and then the adherent cells (BMDMs) were harvested for further experiments. Primary peritoneal macrophages (PEMs) were isolated from the peritoneal cavity of mice.

Human PBMC-derived macrophages were prepared from human peripheral blood using density gradient separation. PBMC was enriched by adherence to plates for 1 h at 37°C in FBS-free RPMI 1640 medium. Then, nonadherent cells were removed by extensive washing with PBS. Monocytes were cultured in complete RPMI 1640 medium containing 10 ng/ml recombinant human M-CSF (PeproTech) to induce macrophages. Human macrophages were harvested for phagocytosis assay on day 7.
To generate mouse M1 macrophages, peritoneal or bone marrow cells were isolated from female Balb/c mice and cultured with 5 ng/mL recombinant mouse GM-CSF (PeproTech) for 7 days. To generate M0 or M2 macrophages, bone marrow cells were isolated from female Balb/c mice and cultured with 25 ng/mL recombinant mouse M-CSF (PeproTech) for 7 days. On Day 5, M1 polarization was achieved with further treatment on day 5 by 20 ng/mL IFN-γ (PeproTech) stimulation for 1 h, followed by 100 ng/mL LPS (Sigma-Aldrich) for 48 h. M2 polarization was achieved by further treatment with 20 ng/mL IL-4 (PeproTech) and 20 ng/mL IL-13 (PeproTech) for 48 h. Macrophages were arrested for phagocytosis assay on day 7.