Interferon gamma ifn γ

U937 cells were induced into macrophages according to reference [20 (link)]. U937 cells were collected by centrifugation and inoculated in a 6-well plate at 8 × 10⁵ cells/well. U937-M0 (inactive type) was obtained using phorbol myristate acetate (PMA, 5 ng/mL, Biyuntian Company). PMA was added in the dark and the cells cultured for 48 h at 37 °C with 5% CO₂. The medium containing PMA was then replaced, and 20 ng/mL interferon-gamma (IFN-γ, PeproTech, Rocky Hill, NJ, USA) and 100 ng/mL lipopolysaccharide (LPS, Sigma, St. Louis, MO, USA) were added for 24 h to induce and achieve U937-M1 cells (classic activated type).

The isolated monocytes were cultured for 5 days in RPMI 1640, supplemented with 10% FBS and 25 ng/ml of Recombinant Macrophage Colony-Stimulating Factor (M-CSF, PeproTech, Rocky Hill, USA) to generate M0 macrophages. After 5 days, macrophages were polarized in vitro toward M1 or M2 phenotypes. For M1-like polarization, macrophages were cultured in RPMI-1640 medium supplemented with 100 ng/ml lipopolysaccharides (LPS from E. coli; Sigma-Aldrich) and 50 ng/ml interferon-gamma (IFN-γ; PeproTech) and incubated for 18 h. Meanwhile, for M2-like polarization macrophages were cultured in a complete RPMI-1640 medium supplemented with 20 ng/ml interleukin-4 (IL-4; PeproTech), for 18 h.
The culture medium of M2 macrophages was supplemented with tumor necrosis factor-alpha (TNFα; 20 ng/ml; PeproTech) and Transforming Growth Factor-beta (TGFβ; 10 ng/ml; PeproTech) for 18 h as previously reported [18 (link)].

Human umbilical vein endothelial cells (HUVECs), primary human dermal fibroblasts (HDFs), and related media for cell culture, including endothelial growth medium, were purchased from Lonza (Walkersville, MD, USA). HDFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F12) (Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS) (FB Essence, VWR, PA, USA) and 1% penicillin/streptomycin (HyClone, IL, USA) and incubated at 37 °C and 5% CO₂ throughout the experiment. THP-1 human monocytic cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI-1640) medium (Corning) supplemented with 10% fetal bovine essence (FBE; VWR, USA) and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, Milwaukee, WI, USA). Phosphate-buffered saline (PBS) and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay were purchased from Promega (Madison, WI, USA), while growth factor reduced matrigel BD was from Corning. The cell staining solution was obtained from Cell Biolabs (San Diego, CA, USA) and Culture-Insert was purchased from IbiTreat (Martinsried, Germany). 12-myristate 13-acetate (PMA) and LPS from Escherichia Coli were purchased from Sigma. Interferon-gamma (IFN-γ) was purchased from PeproTech (Rocky Hill, NJ, USA).

The following reagents were used to treat cells: lipopolysaccharide (LPS) from E. coli strain O127:B8 (100 ng/mL; Sigma Aldrich), Pam₃CSK₄ (100 ng/mL; Invivogen), R-FSL-1 (250 ng/mL; EMC Microcollection Gmbh), interferon gamma (IFNγ; 10 ng/mL; Peprotech) and adenosine triphosphate (ATP; 5 mM; Sigma Aldrich). Concentrations of interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor (TNF) in cell supernatants were measured using the Human Inflammatory Cytokine Cytometric Bead Array Kit (BD Biosciences). Concentrations of chemokines in cell supernatants were measured using the LEGENDplex™ Human Proinflammatory Chemokine Panel 1 (Biolegend). Readings were made on an Attune™ Nxt Flow Cytometer.