Atcc pcs 201 012

Primary human adult dermal fibroblasts (HDFa, ATCC^® PCS-201-012™), mouse macrophages (RAW 264.7), penicillin streptomycin, fetal bovine serum (FBS), phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay kit and Dulbecco’s modified eagle medium (DMEM high glucose 4500 mg/L) were all purchased from ATCC (Manassas, VA, USA). DMEM (no glucose), sodium pyruvate and Hoescht dye were obtained from Fischersci (Hanover Park, IL, USA). Lipopolysaccharide pseudomonas aeroguinosa (LPS), Calcein-AM, protease inhibitor cocktail, mouse nitric oxide synthase and dopamine-HCl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethidium bromide was acquired from Invitrogen (Grand Island, NY, USA). Human recombinant Interferon γ (hrIFN-γ) was purchased from GenScript (Piscataway, NJ, USA). The primary and secondary antibodies against CD90/Thy1 (ab23894) protein and (iNOS) (ab136918) were purchased from Abcam (Cambridge, MA, USA). The 7.5% SDS polyacrylamide gels and gelatin were acquired from Bio-Rad (Hercules, CA, USA). The Odyssey western blot starter kit 2 was obtained from LI-COR (Lincoln, NE, USA). Silicone elastomer base and curing agent (Dow Corning Sylgard^®184) were obtained from ML Solar LLC (Campbell, CA, USA).

Primary human dermal fibroblasts isolated from adult skin (HDFa, ATCC^® PCS-201-012™, Manassas, VA, USA) were cultivated in a supplemented Eagle’s Minimum Essential Medium (EMEM) (10% fetal bovine serum, 2 mM of glutamine and 1% penicillin–streptomycin antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin)) at 37 °C in a humidified atmosphere with 5% CO₂. The hydroalcoholic extract obtained previously (Section 2.2.1) was dried in a vacuum until all solvent was removed and the resulting crude extract (PCext) was resuspended in EMEM, to achieve the final concentration of 100 µg/mL. AAPH (2,2′-azobis(2-amidinopropane) dihydrochloride) (10 mM) diluted in EMEM was used to induce oxidative damage. A total of four experimental groups were formed as follows: (i) Ctrl—cells were incubated with the culture medium only; (ii) PCext—cells were incubated with pechiche crude extract for 24 h; (iii) AAPH—cells were incubated with AAPH (10 mM) for 24 h; and (iv) PCext + AAPH—cells were incubated with PCext for 24 h and then with AAPH (10 mM) for 24 h. The dose/time combination of the AAPH (10 mM) was determined through preliminary cytotoxicity assays and was selected according to its capacity to decrease cell vitality (~50%) compared to the control group (p ≤ 0.05) using the MTT [3-(4,5-diphenyl-tetrazolium bromide] colorimetric assay [16 ].

Hepatocellular carcinoma (HepG2) (ATCC HB-8065™), primary dermal fibroblasts (HDFa) (ATCC PCS-201-012™) and pancreatic islet cell tumor (RIN-m) (ATCC CRL-2058™) cell lines were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA). Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 (DMEM-F12) at pH 7.4, fetal bovine serum (FBS), phosphate-buffered saline (1×) solution at pH 7.4 (PBS) and trypsin 0.25% EDTA (1×) were purchased from Gibco (Grand Island, NY). FITC-labeled human insulin, human recombinant insulin, fluorescein-5-isothiocyanate (FITC), dimethyl sulfoxide (DMSO), D-(+)-glucose (≥95.5%), 3,5-dinitrosalicylic acid (DNS), potassium sodium tartrate tetrahydrate, potassium disulfide and phenol were purchased from Sigma-Aldrich (Saint Louis, MO, USA).