Dulbecco s modified eagle dmem

Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco's modified Eagle (DMEM), Eagle's minimum essential medium (EMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium iodide (PI), trypan blue, trihydroxy benzoic acid (Gallic acids), and Folin–Ciocalteu's reagent were purchased from Sigma (St. Louis, MO, USA). All the plates used in this study were purchased from Corning (Corning, NY, USA). Ethanol, acetone, and hexane were purchased from RCI Labscan (Bangkok, Thai-land). Ammonium persulfate, tetramethyl ethylenediamine (TEMED), protein marker, and acrylamide were purchased from Bio-Rad (California, USA). Tris base was purchased from Vivantis (Selangor Darul Ehsan, Malaysia).

MSCs were obtained from female Sprague Dawley healthy rats. Animals were euthanized with an overdose of pentobarbital sodium. Bone marrow was obtained from both femurs and tibias using a 200 μL micropipette and deposited in a 15 mL conical tube with culture medium [Dulbecco's Modified Eagle [DMEM], from GIBCO]. The sample was centrifuged at 1500 rpm for 7 min. Cells were then separated using a Ficoll (Sigma-Aldrich) (3 mL) gradient centrifuged at 2,000 rpm at 24°C for 30 min. The total number of nucleated cells obtained was quantified and 9 × 10⁶ cells were seeded into a 75 cm² culture flask (in 5 mL of DMEM with 20% fetal bovine serum (FBS), from GIBCO), 1 mL of L-Glutamine (GIBCO), 5 mL of HEPES (Sigma-Aldrich), and 1 mL of Penicillin-Streptomycin (GIBCO). Cells were then placed in a water-jacketed incubator at 37°C with 5% CO2 until they formed a fibroblast monolayer. Finally, MSCs were reseeded onto the fibroblast layer and maintained for 2 weeks until transplantation.