Transforming growth factor β1 tgf β1

Human CRC HCT116 cells were purchased from the American Type Culture Collection (ATCC; CCL-247™; Manassas, VA, USA). The cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Life Technologies), 100 U/ml penicillin–100 μg/ml streptomycin solutions (Gibco, Life Technologies), and 2.5 mg/ml sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) with 5% CO₂. Transforming growth factor-β1 (TGF-β1; 10 ng/ml; Sigma-Aldrich) was used to stimulate the EMT process21 (link).

Peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats of healthy anonymous donors. Buffy coats were provided by the blood donation center of the Justus-Liebig-University Giessen, Germany. Cells from the donors were not pooled. Lymphocytes and monocytes were separated from the blood by density gradient centrifugation based on Ficoll (LeucoSep, greiner bio-one, Frickenhausen, Germany). Subsequently, 10.5 × 10⁵ PBMCs per cm² were seeded into 24-well plates and incubated with DMEM high glucose including 10% fetal calf serum, 100 U/ml penicillin, 100 μg/g streptomycin and 25 ng/ml macrophage-colony stimulating factor (M-CSF) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). To induce osteoclastogenesis, 25 ng/ml receptor activator of NFκB ligand (RANKL) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1 ng/ml transforming growth factor-β1 (TGF-β1) (Sigma-Aldrich) and small pieces of calf bone slices were given to the monocytes after 72 h. Additionally, the corresponding amount of AgNPs was given to the cells. The cells were incubated at 37 °C and 6% CO₂ and the osteoclast medium, including the AgNPs, was changed twice per week.