Dulbecco s modified eagle s medium dmem

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Biowest (Nuaillé, France). Polyvinylidene difluoride (PVDF) membranes and goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin (Ig) G were obtained from Millipore (Bellerica, MA, USA). MTT, 3-MA, salubrinal, Z-DEVD-FMK, Z-VAD-FMK and β-actin antibody were purchased from Sigma (St. Louis, MO, USA). An annexin V-FITC/PI apoptosis detection kit was purchased from Pharmingen (San Diego, CA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from Pierce Biotechnology (Rockford, IL, USA).

All reagents used in the experiment were guaranteed reagent grade and HPLC-grade. Acetonitrile, ethanol and methanol were from Merck (Darmstadt, Germany). Isopropanol (100%) was from J.T. Baker Chemical (Phillipsburg, NJ, USA). Phosphate-buffered saline (PBS) was from Lonza (Walkersville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from BioWest (Riverside, MO, USA). Paraformaldehyde (4%) was from Biosesang (Seongnam-si, Korea). Bovine calf serum (BCS), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and insulin were from Gibco (Grand Island, NY, USA). Ginsenoside Rg₁, Rb₁ and Rg₃(S) were from ChromaDex Co. (Irvine, CA, USA). Dexamethasone (Dex), 3-isobutyl-1-methylxanthine (IBMX), thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Oil Red O, glycyrrhizin, 2-methyl-2-butanol and 2,2,2-tribromoethanol (Avertin) were from Sigma-Aldrich (St. Louis, MO, USA). RIPA buffer and bicinchoninic acid (BCA) protein assay kit were from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies, anti-rabbit β-actin, PPARγ, C/EBPα, adiponectin, AMPK, p-AMPK, ACC, p-ACC were from Cell Signaling Technology (Danvers, MA, USA), SREBP-1c and CPT-1 from Santa Cruz Biotechnology (Dallas, TX, USA).

Chitosan (CHT) with viscosity molecular weight of 223,332 gmol⁻¹, (determined experimentally [24 (link)]) and a degree of deacetylation of 84.1% (Batch number: STBF4197V provided by the supplier), polyethylene glycol diglycidyl ether (Mn 500) and glutaraldehyde (GA) solution Grade II, 25% in H₂O were purchased from Sigma-Aldrich (Saint Louis, MO, USA ), while J.T. Baker provided acetic acid (Xalostoc, México). MTS CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation obtained from Promega (Madison, WI, USA) Dulbecco’s modified Eagle’s medium (DMEM) from Biowest (Riverside, MO, USA), human osteoblast hFOB1.19 from ATCC^® CRL-11372™ (Manassas, VA, USA). Integrin β1 (A-4): sc-374429, vinculin (7F9) sc-73614 and antibody (normal mouse IgG: sc-3891 SCBT) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Human gingival fibroblasts (HGFs; IBRC C10459) purchased from the Iranian Biological Resource Center (Tehran, Iran) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biowest, France) supplemented with 10% fetal bovine serum (Gibco, UK) and pen-streptomycin (Biowest, France). The cells were seeded into the 96-well microtiterplate at a density of 10,000 cells/well followed by overnight incubation. Parallel to this experiment, a range of concentrations from 78.1 to 625 μg/mL of CS, ZnONC and ZnONC-CS were shaken in an incubator for 24, 48, and 72 h in DMEM to prepare extraction media. The seeded medium was replaced with 200 μL of the extraction media followed by incubation for 24 h. Post incubation, the cells were washed with fresh sterile phosphate-buffered saline (PBS) to eliminate non-adherent cells and media. Finally, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Sigma-Aldrich) was used to determine the cytotoxicity in HGF cells at 570 nm according to the manufacturer’s instructions [34 (link)]. Dimethyl sulfoxide (DMSO) at 10% concentration served as the cell death control (positive control). The permissible limit of cytotoxicity effect is considered to be > 75% according to ISO standards 10,993–5:2009 [35 ].

Keloid tissues were obtained from six patients who underwent excision surgery at Seoul National University Boramae Hospital. The keloid tissues were washed three times with Dulbecco’s phosphate-buffered saline with 1% antibiotic–antimycotic solution (A/A; Welgene, Gyeongsangbuk-do, Republic of Korea) and cut into 3-mm-thick pieces. Subsequently, the tissues were digested with 5 mg/ml dispase (Roche, Basel, Switzerland) for 4 h at 37 °C. The dermis and epidermis were separated, cut into 1-mm-thick pieces, and digested to a single-cell suspension with 3 mg/ml collagenase type I (Thermo Fisher, Waltham, MA, USA). The cells were growth in Dulbecco’s modified Eagle’s medium (DMEM; Biowest, Nuaillé, France) containing 10% fetal bovine serum (FBS; Biowest, France) and 1% A/A in a humidified incubator with 5% CO₂ at 37 °C. Cells from passage 3 to 5 were used for experiments.