L glutathione oxidized

Purpurin (purity ≥ 85.0%), Curcumin (purity ≥ 98.0%), L-glutathione reduced (GSH, purity ≥ 98.0%), L-glutathione oxidized (GSSG, purity ≥ 98.0%), L-buthionine-sulfoximine (BSO, purity ≥ 97.0%), N-acetyl-L-cysteine (NAC, purity ≥ 99.0%), tert-butyl hydroperoxide (TBHP, 70% in water) 2′,7′-dichlorofluorescein (DCF, purity ~90%) and 2′,7′-dichlorofluorescein diacetate (H2DCF-DA, purity ≥ 97.0%) were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands). All buffer salts, including citric acid, tri-sodium citrate dihydrate, di-sodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate and formic acid, and acetonitrile for Liquid Chromatography/Mass Spectrometry assay, were also obtained from Sigma Aldrich (Zwijndrecht, The Netherlands). Methanol of analytical grade was ordered from Fisher Scientific (Loughborough, United Kingdom). Dimethyl sulfoxide (DMSO) was purchased from Acros Organics (Geel, Belgium). Dulbecco’s Modified Eagle Medium with Ham’s Nutrient Mixture F-12 (1:1) (DMEM/F12), DMEM/F12 without phenol red, trypsin 0.05% EDTA, nonessential amino acids (NEAA) and phosphate-buffered saline pH 7.4 (PBS), geneticin (G418), penicillin/streptomycin, were purchased from Gibco (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Invitrogen (Breda, The Netherlands).

Drugs and reagents were obtained from the following sources: 3-(3-carbamoylphenyl)phenyl N-cyclohexylcarbamate (URB597), 4-hyroxynonenal (4-HNE), 8-iso Prostaglandin F_2α-d₄ (8-isoPGF2α–d₄), 8-iso Prostaglandin F_2α (8-isoPGF2α) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) from Cayman Chemical Company (Ann Arbor, MI, USA); 11-deoxycorticosterone acetate (DOCA), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), xanthine, xanthine oxidase (XO), 9,9′-Bis(N-methylacridinium nitrate) (lucigenin), superoxide dismutase (SOD), catalase (CAT) thioredoxin (Trx), l-ascrobic acid, retinol, l-glutathione reduced (GSH), l-glutathione oxidized (GSSG), benzaldehyde-d₆ and Tween 80 were acquired from Sigma-Aldrich (Steinheim, Germany); pentobarbital sodium was purchased from Biowet (Puławy, Poland); chloro-2,4-dinitro benzene (CDNB), butylated hydroxytoluene (BHT), and 5,5′-dithiobis (2-dinitrobenzoic acid) (DTNB) were acquired from Sigma-Aldrich (Steinheim, Germany). DOCA was dissolved in DMF, whereas URB597 was dissolved in an URB597 solvent: a mixture of DMSO, Tween 80, and saline (0.9% NaCl) [1:2:7; v/v/v].

l-Glutathione reduced (GSH, powder ≥98.0%, m.w. 307.32 g/mol), l-Glutathione oxidized (GSSG, ≥98.0%), 1,4-Naphthoquinone (NPQ, 97%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Catalase from bovine liver (CAT, lyophilized powder, 2000–5000 units/mg) were purchased from Sigma Aldrich (Steinheim, Germany). SLMs were prepared with Dynasan 114 or trimyristin (C14) and Dynasan 118 or tristearin (C18), which were obtained from Sasol (Witten, Germany). The colon cancer cell line HT29 was purchased from the American Type Culture Collection (Manassas, VA, USA). For cell culture, RPMI 1640 medium was obtained from Labtek Eurobio (Milan, Italy), fetal calf serum from Euroclone (Milan, Italy) and glutamine, methylthiazolyldiphenyl-tetrazolium bromide (MTT) and 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) were purchesed from Sigma-Aldrich (St. Louis, MO, USA).

Formic acid, HPLC grade methanol, acetone, hydrochloric acid, trifluoroacetic acid, ammonium formiate, L-ascorbic acid, and phloroglucinol were purchased from Sigma Aldrich (St Louis, MO, USA). Deionized water was obtained from a Milli-Q purification system (Millipore, Molsheim, France). Standards of (+)-catechin, (–)-epicatechin, (–)-epicatechin 3-O-gallate, reduced L-glutathione, oxidized L-glutathione, piceatannol, p-coumaric acid, protocatechuic acid, syringic acid, trans-caftaric acid, and trans-resveratrol were purchased from Sigma-Aldrich (St Louis, MO, USA). Standards of (–)-epigallocatechin, gallic acid, hydroxytyrosol, malvidin 3-O-glucoside chloride, malvidin 3,5-di-O-glucoside chloride, procyanidin B2, quercetin 3-O-glucuronide, and taxifolin were purchased from Extrasynthese (Geney, France). Standards of caffeic acid, ferulic acid, and vanillic acid were purchased from Fluka (Buchs, Switzerland). Standards of 1-O-Galloyl-β-D-glucose and quercetin 3-O-glucoside were purchased from PlantMetaChem, Transmit GmbH (Gießen, Germany). Standard of trans-piceid was purchased from Selleckchem (Houston, TX, USA).

To extract proteins, isolated primary tumors and spleens were homogenized and lysed with lysis buffer supplemented with protease inhibitors [1% Nonidet P-40, 10 mM Tris-HCl at pH 7.4, 200 mM NaCl, 5 mM EDTA, 10% glycerol, 100 μM phenylmethylsulfonyl (PMSF), 1 mM oxidized L-glutathione (all from Sigma-Aldrich), 0.15 μM aprotinin and 2.1 μM leupeptin (Roche, Mannheim, Germany)] as previously described (4 (link)). Serum was derived from blood which was collected through cardiac puncture, allowed to clot at 37°C for 30 min and centrifuged at 17,000 g for 1 h at 4°C. The levels of 10 selected cytokines (BAFF, G-CSF, IFN-γ, IL-1β, IL-4, IL-6, IL-10, MCP-1, MIP-2, and TNF-α) were measured through Luminex Multiplex Assays (Thermo Fisher Scientific) in primary tumor lysates (50 μg of protein) and sera (1:4 diluted in assay diluent). Enzyme-linked immunosorbent assay (ELISA) was used to measure CHI3L1 and LCN2 levels (Mouse Quantikine ELISA Kit, Biotechne, Minneapolis, MN, USA) in primary tumor lysates, sera and spleen lysates, and the levels of MMP-9, VEGF (Mouse Quantikine ELISA Kit, Biotechne) and TGF-β1 (Mouse uncoated ELISA Kit, Thermo Fisher Scientific) in primary tumor lysates and sera with absorbances at 550 nm subtracted from those at 450 nm for correction. ELISA data were analyzed by means of Deltasoft JV (BioMetallics Incorporated).