Murine raw264.7 macrophages

Murine RAW264.7 macrophages were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning, Tewksbury, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Corning) and 1% penicillin and streptomycin (Corning). Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. LPS (List Labs, Campbell, CA, USA) was dissolved in distilled water to a stock concentration of 1 mg/mL and further diluted to 100 ng/mL in cell culture media.

Murine RAW264.7 macrophages obtained from the American Type Culture Collection (ATCC, VA, United States), were cultured in complete RPMI 1640 containing 10% heated-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ in air.

Murine RAW264.7 macrophages were obtained from the American Type Culture Collection (ATCC; Manassas, VI, USA). Cells were cultivated in 150 cm² cell culture flasks (TPP Techno Plastic Products, Trasadingen, Switzerland) in high-glucose (4500 mg/L) Dulbecco’s Modified Eagle Medium (DMEM) containing 0.1 mg/mL penicillin/streptomycin/L-glutamine (PSG) and 10% (v/v) FBS. As recommended by the ATCC, cells were sub-cultured at about 80% confluence by detaching with a cell scraper and culturing with a mixture of 70% high-glucose DMEM (supplemented with PSG and FBS) and 30% culture medium, which was obtained from the previous passage [4 (link)]. Cells were kept at 37 °C in a humidified 5% CO₂/95% (v/v) air atmosphere. For further experiments, harvested cells were seeded at different densities, cultured for another 24 h, and incubated with compounds for the times indicated.

Primary BMDM were prepared from C57BL/6 mice, MerTK^−/− mice, STAT1^−/− mice, and WT mice as described previously48 (link). Briefly, BMDM were differentiated from murine bone marrow myeloid stem cells. Bone marrow cells were cultured for 7–10 days with DMEM supplemented with 10% L929 supernatant containing 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, and 5 × 10⁻⁵ M 2-mercaptoethanol. BMDM differentiation was confirmed by FACS analysis using anti-CD11b. Murine RAW 264.7 macrophages [American Type Culture Collection (ATCC), Manassas, VA, USA] were plated at a density of 10⁶ cells/ml and incubated overnight in Dulbecco’s modified Eagle’s medium (DMEM, Media Tech Inc., Washington, DC, USA) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO₂. Prior to stimulation, the medium was replaced with serum-free X-VIVO medium (Lonza, Basel, Switzerland). On the other hand, BMDC were induced to differentiate by the addition of recombinant mouse GM-CSF (rmGM-CSF, R&D Systems) (20 ng/ml) every 3 days and LPS (1 μl/ml, Sigma-Aldrich) on day 10. After another 48 h, cells and medium were collected for further assays.

Murine RAW264.7 macrophages and 4T1 and EMT6 murine mammary carcinoma cells were purchased from American Type Culture Collection (ATCC). Human embryonic kidney (HEK293) cells were obtained from Prof. D. Joseph Jerry (Veterinary and Animal Sciences, UMass Amherst). All cell lines, including RAW:iNos-eGFP, were cultured at 37°C under a humidified atmosphere containing 5% CO₂. Standard growth media consisted of high glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Corning), 1% L-Glutamine (200 mM, Gibco) and 1% antibiotics (100 μg/ml penicillin and 100 μg/ml streptomycin, Gibco) – herein referred to as complete DMEM. Under the above culture conditions the cells were sub-cultured approximately once every 3-4 days and only cells between passages 7 and 20 were used for all experiments.