5 5 dithiobis 2 nitrobenzoic acid dtnb thiobarbituric acid tba

MeDHICA was prepared as described in [42 (link)]. MeDHICA-melanin was prepared by aerial oxidation of MeDHICA in phosphate buffer at pH 8.5, as previously reported [42 (link)]. Phosphate buffer saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (HyClone), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), l-glutamine, trypsin-EDTA, Triton, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), thiobarbituric acid (TBA), and Bradford reagent were from Sigma-Aldrich (St. Louis, MI, USA). Bicinchoninic acid (BCA) protein assay kit was from Thermo Scientific (Waltham, MA, USA). Antibodies against nuclear factor erythroid 2–related factor 2 (Nrf-2) and heme oxygenase 1 (HO-1) were from Cell Signal Technology (Danvers, MA, USA). Antibodies against B-23 and β-actin and the chemiluminescence detection system (SuperSignal^® West Pico) were from Thermo Fisher Scientific (Waltham, MA, USA).

CORM-2, 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), thiobarbituric acid (TBA) and H₂O₂ were purchased from Sigma Chemical Co. (Germany). The coagulation reagents were obtained from Diagon Ltd. (Hungary) and Behringer Ingelheim. All other chemicals were reagent-grade products purchased from POCh (Gliwice, Poland).
A stock solution of CORM-2 was made in DMSO (the final concentration of DMSO in a stock solution of CORM-2 was 50%). The final concentration of DMSO in samples was lower than 0.05% and its effects were determined in all experiments. Inactive CORM-2 (i-CORM-2) was prepared by keeping the stock solution in 50% DMSO over night at 37°C [13 (link),14 ].