2 2 azinobis 3 ethylbenzthiazoline 6 sulfonic acid

WPI (protein content > 91.5%) was acquired from Hilmar Industries (Hilmar, CA, USA). Thioflavine T (ThT), 8-anilino-1-naphthalenesulfonic acid (ANS), Congo red, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents were higher than analytical grade.

Arbutin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), l-tyrosine, l-DOPA (l-3,4-dihydroxy phenylalanine), ascorbic acid, Tween-20, thiazolyl blue tetrazolium bromide (MTT), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), O-tetradecanoyl phorbol-13-acetate (TPA), mushroom tyrosinase, 6-hydrolxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals and reagents were high quality-grade and commercially obtainable. Antibodies purchased from Bioworld Technology (St. Louis Park, MN, USA) included anti-Tyr (Cat. No. BS6754), anti-TyrP-1 (Cat. No. sc-25543), anti-TyrP-2 (Cat. No. BS3320), anti-MITF (Cat. No. BS1550 for Western blotting), anti-phospho-JNK (Cat. No. 4668), anti-JNK (Cat. No. 9252), anti-phospho-p38 (Cat. No. 4511), anti-p38 (Cat. No. 9212), anti-p44/42MAPK (ERK1/2; Cat. No. 9102). Anti-phospho-p44/42MAPK (ERK1/2; Cat. No. 4376) and anti-MITF (Cat. No. 12590 for signaling) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Cytokines were measured in the supernatants of peritoneal exudates after VTn-challenge by a specific two-site sandwich ELISA using antibody pairs purchased from Pharmingen (BD, San Diego, CA, USA). Binding of biotinylated monoclonal antibodies was detected using streptoavidin-horseradish peroxidase complex (Amersham Int., Amersham, UK) and 2-2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma) in 0.1 M citrate buffer containing hydrogen peroxide. Samples were quantified by comparison with standard curves of recombinant mouse cytokines. Detection limits were 3.9 ng/mL for IL-4, 4.21 ng/mL for IL-5, 1.21 pg/mL for IL-17A, 2.56 ng/mL for IFN-γ, and 1.71 ng/mL for eotaxin.

Compounds which are used for antioxidant activity suchlike neocuproine (2,9-dimethyl-1,10-phenanthroline), 2,2-azino-bis3-ethylbenzthiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (Ferrozine), ascorbic acid, BHT (butylated hydroxytoluene), α-tocopherol were obtained from Sigma (Sigma-Aldrich GmbH, Steinheim, Germany). All other chemicals used were of analytical grade and obtained from either Sigma-Aldrich or Merck Millipore.

OVA-specific IgE, IgG₁, and IgG_2a antibody titers in sera were determined by ELISA (Becton Dickinson Biosciences, San Jose, CA, USA) as described previously [13 (link)]. In brief, 96-well microtiter plates were coated with OVA (10 μg/mL) in NaHCO₃ buffer (pH 9.6) at 4°C overnight, and then treated with mouse sera followed by biotin-conjugated anti-mouse IgE, IgG₁, or IgG_2a (PharMingen, San Diego, CA) for 1 h at 37°C. Then, avidin-horseradish peroxidase (1 : 5000, Pierce Biotechnology, Rockford, IL, USA) was added to each well for another 30 min at room temperature. Finally, the reaction was developed by 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich, St. Louis, MO) and the absorbance was determined at 405 nm. The levels of antibody were compared with OVA-specific IgE, IgG₁ and IgG_2a standard serum with predetermined concentrations (immunoglobulin concentrations: IgE = 1.5 μg/mL, IgG₁ = 32.5 μg/mL IgG_2a = 15.2 μg/mL). The concentration of standard serum was arbitrarily assigned as 1 ELISA unit (1 EU).