Trypsin edta solution 0.25

Primary human dermal fibroblasts (HDF), human breast cancer cell line MDA-MB-231 (MDA), human cervix adenocarcinoma cells (HeLa), and hypopharyngeal carcinoma cells (FaDu) cells were purchased from ATCC^®. HDF, HeLa, and FaDu cells were cultured and maintained using Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich^®) containing: 10 (v/v) fetal calf serum, 2 mM L-glutamine, 100 mg/ml streptomycin and 100 IU/ml penicillin (Sigma-Aldrich^®). Cells were cultured at 37°C/95% air/5% CO₂. Human umbilical vein endothelial cells (HUVECs) were purchased from Life Technologies^™ and cultured using Medium 200 with Low Serum Growth Supplement (LSGS) (Life Technologies^™, USA) and maintained as reported for the previous cells. MDA-MB-231 cells were cultured in RPMI 1640 medium (Lonza^®), containing: 10 (v/v) fetal calf serum, 2 mM L-glutamine, 100 mg/ml streptomycin and 100 IU/ml penicillin (Sigma-Aldrich^®). Cells were periodically sub-cultured using Trypsin-EDTA solution 0.25% (Sigma-Aldrich^®) for the detachment process and centrifuged at 2000 rpm for 5 min for the pellet collection.
The cellular synchronisation was obtained by incubating the cells with serum-deprived medium, at different time points (18, 24, 36 and 48 hours), depending on the experimental conditions.

Cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ in 25 mL bottles for a maximum of 30 days.
Once the cultures reached 70–80% confluency, they were passaged by washing with 3 mL of D-PBS, digested with 3 mL of 0.025% trypsin/EDTA (Trypsin-EDTA solution (0.25%), SIGMA-ALDRICH, St. Louis, MO, USA, Merck KGaA, Darmstadt, Germany) at 37 °C for 3–5 min, neutralized by the addition of 6 mL of DMEM supplemented with 10% FBS, 4 mM L-glutamine solution (L-glutamine solution (200 mM), SIGMA-ALDRICH, St. Louis, MO, USA, Merck KGaA, Darmstadt, Germany) and 1% antibiotics/antimycotics (100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 0.25 μg/mL amphotericin B) (Antibiotic Antimycotic Solution (100×); SIGMA-ALDRICH, St. Louis, MO, USA, Merck KGaA, Darmstadt, Germany), centrifuged (5 min, 200× g, room temperature), and resuspended in DMEM/F12 supplemented (cardiomyocytes culture medium).
The medium was changed every 3 days and the culture was observed under an inverted microscope employing relief contrast (IX73, Olympus, Tokyo, Japan) every day. Before the harvesting of cells for further experiments, photos of the culture at three time points, 7 days, 15 days, and 30 days, were also taken to observe possible morphological changes.

All chemicals used in this assay were of analytical grade. Absolute ethanol was provided by Point of Care Diagnostics (North Rocks, NSW, Australia). Ascorbic acid was purchased from Merck (Darmstadt, Germany). 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), an α-glucosidase enzyme (EC-No.: 232-604-7) from Saccharomyces cerevisiae (lyophilised powder, 23 units/mg), 4-nitrophenyl α-D-glucopyranoside (p-NPG, ≥99%), acarbose (99%), Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM), bovine calf serum (BCS), penicillin, streptomycin, glutamine (PSG), fetal bovine serum (FBS), rosiglitazone, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), insulin, phosphate-buffered saline (PBS), (3-(4,5 dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide) (MTT), trypsin-EDTA solution 0.25%, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 3T3-L1 murine cell line was supplied by American Type Tissue Culture/ATCC (Manassas, VA, USA).

CS (degree of deacetylation (DDA) 90.28% and molecular weight (MW) 50–200 kDa) was purchased from MP Biomedicals (Eschwege, Germany), HA, R-Alpha-LA, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS), GSH, Tween 20, Trypsin/EDTA solution 0.25%, rabbit polyclonal antibodies (Abs): CD44, and MTT were provided from Sigma–Aldrich (St. Louis, MO, USA). Fetal Bovine Serum (FBS), L-glutamine (200 mM), and penicillin-streptomycin (10000 U/mL) were obtained from Biochrom (Berlin, Germany). Dead-end Fluorometric TUNEL System and Cell Titer 96^® Aqueous One Solution Cell Proliferation Assay were prepared from Promega (Madison, USA). MCF-7 and BT-20 cell lines were supplied from Pasteur Institute (Tehran, Iran) and MT from Aburaihan Pharmaceutical (Tehran, Iran). Dulbecco’s Modified Eagle Medium (DMEM) and MEM media, Fluorescein isothiocyanate (FITC)-conjugated (green) secondary Ab, and anti-fade reagent were obtained from GIBCO Invitrogen (Grand Island, NY). Kits of LDH cytotoxicity, Caspase-3 Fluorescence assay, and JC-1 Mitochondrial Membrane Potential assay were supplied from (Cayman Chemical, Michigan, MI, USA). All of the other chemicals and solvents were purchased from Merck (Darmstadt, Germany).

All solvents, reagents, and standards used in this study were of analytical grade. Vanillic, caffeic, ferulic, p-coumaric acids and vanillin, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA), penicillin-streptomycin solution, trypsin-EDTA solution 0.25% were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). HPLC-grade acetonitrile, trifluoroacetic acid (TFA) and acetic acid (glacial) were obtained from Sigma-Aldrich GmbH (Buchs, Switzerland). Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax, fetal bovine serum were obtained from Gibco (UK); MitoSOX and Mitotracker Green were purchased from Invitrogen (Eugene, USA).