12 myristate 13 acetate pma

Human monocytic cells (THP-1 cells) were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS), and 0.05 mM 2-mercaptoethanol. THP-1 cells were differentiated into M0 macrophages by culturing the cells with 100 ng/ml of 12-myristate 13-acetate (PMA) (Sigma) for 24 hours^{17 ,80 (link),81 (link)}. After differentiation, the cells were washed three times with serum free culture media to remove non-differentiated cells. To activate M0 macrophages, the cells were exposed to polarization media, which consisted of culture media supplemented with 100 ng/ml of lipopolysaccharide (LPS) (Sigma) from Escherichia Coli serotype 0111:B4 and 20 ng/ml of interferon (IFN)-γ (Peprotech). To assess the effects of NAM, RSV, and the combination of RSV and NAM (RSV + NAM), pro-inflammatory macrophages were exposed to RSV (10 µM), NAM (500 µM) or RSV (10 µM) plus NAM (500 µM) for durations of 16, 24, 48, and 120 hours.

All chemical reagents used for synthesis of silver nanoparticles (silver nitrate, tannic acid, and ammonia (25 wt% NH₃) were commercial products of Sigma-Aldrich and Avantor Performance Materials Poland S.A. (formerly POCH S.A.). Natural ruby mica sheets obtained from Continental Trade were used as a substrate for nanoparticle deposition. The pieces of mica were freshly cleaved into thin fragments of desired area. In order to promote silver particle deposition, the cationic polyelectrolyte, poly(allylamine hydrochloride) (PAH), having a molecular weight of 70 kDa (Polysciences) was used for modification of mica surface. Ultrapure water used throughout these investigations was obtained using the Milli-Q Elix&Simplicity 185 purification system from Millipore SA Molsheim, France. Dimethyl sulfoxide (DMSO) and 12-myristate-13-acetate (PMA) and all standard chemicals were purchased from Sigma (USA). RPMI 1640 medium, fetal bovine serum (FBS), and antibiotics were from CytoGen GmbH (Germany).

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).