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|NPM was sterilized by ultraviolet light to kill any possible pathogens in the air sample. NPM-protein corona (NPC) samples were prepared as described above and dissolved in the medium by sonication. HLFs (ATCC® PCS-201-013™) were routinely grown using FGM™ fibroblast growth media (Lonza, USA) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Cells were suspended at 2 × 106 cells/ml and mixed with the NPC solutions. NPM concentrations of NPC-HLFs were fixed at 25, 50 and 100 μg/ml, respectively. To demonstrate the bioactivity of the protein corona, transforming growth factor beta 1 (Sigma, USA), NPC-vitamin C, pure serum and pure nanoscale PM2.5 (NPM) were also mixed with HLFs following the same procedure described above. The final exposure concentrations were 10 ng/ml (TGF-β1), 0.5 mg/ml (vitamin C) and 100 μg/ml (NPM).|
| Cells were maintained in culture medium, DMEM (Wako, Tokyo, Japan) that was supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), and 0.1 mg/ml streptomycin and 100 U/ml penicillin (Meiji Seika, Tokyo, Japan) at 37°C in a humidified atmosphere (5% CO2).
the human lung adenocarcinoma epithelial cell line, A549 (European Collection of Cell Cultures, Salisbury, United Kingdom) was used to measure TGF-β1–induced decreases in E-cadherin levels, a marker of epithelial cells, in the absence or presence of a designated agent.
|A modified Boyden chamber (8‐μm pore filter, 24‐well cell clusters) (Chemotaxicell; Kurabo, Osaka, Japan) coated with type I collagen (Nitta Gelatin, Inc., Osaka, Japan) were used for the chemotaxis assay (Suganuma et al. 2012; Aso et al. 2013). Fibroblasts were cultured on polyacrylamide gels or plastic dishes coated with 10 μg/mL type I collagen in DMEM/F‐12 cell culture medium (Thermo Fisher Scientific) containing 10% FBS for 4 days. The cells were brought to a quiescent state overnight by incubation in DMEM/F‐12 containing 0.1% FBS.|