2 7 dichlorodihydrofluorescein diacetate dcfh2 da

Ellagic acid, galvinoxyl, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and cumene hydroperoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH₂-DA) was obtained from Molecular Probes (Eugene, OR, USA). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and sterile plastic ware for cell culture were procured from Flow Laboratory (Irvine, UK). Fetal bovine serum was acquired from GIBCO (Grand Island, NY, USA). NADPH, ADP and trichloroacetic acid (TCA) were obtained from Sisco Research Laboratory (Mumbai, India). Tris, FeCl₃, thiobarbituric acid (TBA), dimethylsulphoxide (DMSO) of high purity were procured from local suppliers. Ellagic acid peracetate (EAPA) was synthesized and characterized at the Department of Chemistry of the University of Delhi, as previously described [26 (link)].

All chemicals used were of analytical grade. Solvents and reagents were purchased from Sigma Aldrich (now Merck, Dusseldorf, Germany). Acyl chlorides 5a–5j were distilled before use. HTyr 1 [31 (link)] and 2-(2-methoxy-benzo[1,3]dioxol-5-yl)-ethanol 3 were synthesized as described in the literature [27 (link)]. silica gel 60 F254 plates and silica gel 60 were purchased from Merck (Milan, Italy). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH₂-DA) was obtained from Molecular Probes (Eugene, OR, USA). L6 rat skeletal myoblasts and THP-1 monocytes were from the American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and sterile plasticware were from Flow Laboratory (Irvine, CA, USA). Fetal bovine serum was from GIBCO (Grand Island, NY, USA).

Oxidative stress levels were assessed through the redox imbalance between the ROS levels and antioxidant defenses, namely reduced glutathione (GSH), as previously described [38 (link)]. Intracellular peroxides and superoxide anion were measured using the dyes 2,7-dichlorodihydrofluorescein diacetate (DCFH₂-DA; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) and dihydroethidium (DHE; Molecular Probes, Thermo Fisher Scientific), respectively. A quantity of 1 × 10⁶ untreated and parthenolide-treated cells was incubated with 5 µM of DCFH₂-DA for 45 min at 37 °C or 5 µM of DHE for 15 min in the dark at RT.
Mercury orange (MO) dye (Sigma-Aldrich) was used to measure the GSH content. Briefly, 1 × 10⁶ cells were incubated with 40 µM of MO for 15 min in the dark at RT. Cells were washed twice with cold PBS by centrifugation at 300× g for 5 min and resuspended in the same buffer. Flow cytometry was then performed using a FACSCalibur flow cytometer (Becton Dickinson).
At least 25,000 events were acquired using CellQuest software (Becton Dickinson) and analyzed using Paint-a-Gate (Becton Dickinson). The results were presented using the probe’s mean fluorescence intensity (MFI) in each condition.

DMSO (anhydrous, ≥99.9%), tert-butyl ammonium bromide (TBMA), [(2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedionato)silver(I)] and paraquat were purchased from Sigma-Aldrich (St. Louis, MO). Roswell Park Memorial Institute medium (RPMI 1640), Dulbecco´s modified Eagle medium (DMEM), sodium pyruvate (100 mM) streptomycin (100 mg/ml), penicillin (100 U/ml), phosphate buffered saline (PBS: 10 mM Na₂HPO₄, 137 mM NaCl 2,7 mM KCl dissolved in 500 ml of distilled water, pH 7.4). D-glucose (5 mM), dimethyl sulfoxide (DMSO), propidium iodide, Galvinoxyl, cumene hydroperoxide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate buffered saline (PBS)- one tablet/L buffer without Calcium and Magnesium were purchased from Sigma-Aldrich; 2',7'-dichlorodihydrofluorescein diacetate (DCFH₂-DA) was obtained from Molecular Probes (Eugene, OR). Sterile plasticware for cell culture was from Falcon Brand, San Diego (CA), fetal bovine serum was from GIBCO (Grand Island, NY).

All chemicals (analytical grade), silica gel 60 F254 plates, and silica gel 60 were purchased from Sigma-Aldrich (Milan, Italy). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH₂-DA) was supplied by Molecular Probes (Eugene, OR, USA). Fresh pure hydroxytyrosol was synthetized in our laboratory as previously reported [37 (link)]. L6 myoblasts from rat skeletal muscle and THP-1 human leukemic monocytes were purchased from the American Type Culture Collection (Rockville, MD, USA). DMEM (Dulbecco’s modified Eagle’s medium), RPMI 1640, streptomycin (100 mg/mL), penicillin (100 U/mL), d-glucose, and sterile plasticware for cell culture were from Falcon (San Diego, CA, USA). Fetal bovine serum was from GIBCO (Grand Island, NY, USA).

Dulbecco's modified Eagle's medium (DMEM), penicillin and streptomycin were purchased from Gibco, Life Technologies Italia, Milan, Italy. Octa was furnished by Giuliani Pharma, Milan, Italy. Crystalline 8-methoxypsoralen (8-MOP), dimethylsulfoxide (DMSO), 3-(4,5 dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT), butylated hydroxytoluene (BHT), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), N-ethylmaleimide (NEM), thiosalicylic acid (TSA), sodium methoxide, potassium hydroxide (KOH), retinol (ReOH) and all-trans retinoic acid AtRA were from Sigma-Aldrich, Milan, Italy. 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA) was from Molecular Probes (Eugene, OR, USA). All organic solvents used were of HPLC-grade.

Embelin, (Indofine Chemicals, NJ, USA). Dimethyl sulfoxide (DMSO, anhydrous, ≥99.9%), tert-butyl ammonium bromide (TBMA), [(2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedionato)silver(I)] (Sigma-Aldrich, St. Louis, MO, USA). Roswell Park Memorial Institute medium (RPMI 1640), Dulbecco’s modified Eagle medium (DMEM), streptomycin (100 mg/mL), penicillin (100 U/mL), D-glucose, cumene hydroperoxide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate buffered saline (PBS)- one tablet/L buffer without Calcium and Magnesium (Sigma-Aldrich, St. Louis, MO, USA). 2’,7’-dichlorodihydrofluorescein diacetate (DCFH2-DA) (Molecular Probes, Eugene, OR, USA). Sterile plasticware for cell culture (Falcon Brand, San Diego, CA, USA); fetal bovine serum (GIBCO, Grand Island, NY, USA).