2 7 dichlorodihydrofluorescin diacetate dcfh da

The antioxidant activity was assessed in the plasma samples obtained before and after platelet aggregation, and subsequent removal of platelets and aggregates via centrifugation. The fluorometric assay was performed using the 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA; Sigma-Aldrich S.r.l., Milan, Italy) as probe ad 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH, Sigma-Aldrich S.r.l., Milan, Italy) as the radical generator. DCFH was prepared from DCFH-DA via basic hydrolysis: 500 µL of 1 mM DCFH-DA was mixed at 4 °C with 2 mL 0.01 M NaOH while protected from the light. After 20 min, the mixture was neutralized with 2 mL 0.01 M HCl and diluted with phosphate-buffered saline (PBS; 10 µM final concentration). An amount of 20 µL of sample was loaded in triplicate in a 96-well black plate (Corning Incorporated Costar, Euroclone S.p.A., Milan, Italy) and diluted with 50 µL PBS; 20 µL AAPH (10 mM final concentration) and 10 µL DCFH (10 µM final concentration) were added. Oxidation of DCFH to 2′,7′-dichlorofluorescin (DCF) was monitored at 37 °C, setting the excitation at λ 485 nm and emission at λ 535 nm.

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), fluorescein sodium salt, 2,2′-azobis [2 -methylpropionamide] dihydrochloride (AAPH), quercetin dihydrate, 2′-7′-dichlorodihydrofluorescin diacetate (DCFH-DA), were purchased from Sigma-Aldrich (Milan, Italy). Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose culture media, penicillin, streptomycin, L-glutamine, trypan blue, trypsin, were from culture grade and also purchased from Sigma-Aldrich. Fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (PBS) without Mg²⁺ and Ca²⁺ and Hank’s balanced salts solution (HBSS) from culture grade were purchased from Euroclone SpA (Milan, Italy). Methanol was purchased from Carlo Erba SpA (Milan, Italy). Water and acetonitrile were obtained from J. T. Baker (Phillipsburg, NJ, USA). A Common Steviol Glycosides Standards Kit (steviolbioside, dulcoside A, stevioside, rebaudioside A, B, and C) was purchased from Chromadex (LGC Standards S.r.L., Milan, Italy). All other chemicals were from analytical high-performance liquid chromatography (HPLC) grade and purchased from common sources. All solvents and water were accurately degassed before being used in the analyses.

Velvet antler from sika deer (Cervus nippon) was provided by the Zuojia Sika Deer Farm (Jilin, China). The preparation and composition of velvet antler methanol extracts (MEs) were previously described [7 (link)]. Simply, 7 g of antler velvet powder was mixed with 210 mL methanol and was refluxed at 80°C for 1 h. The supernatant was obtained after centrifugation at 8000 g for 15 min. The methanol solvent was removed under vacuum with a rotary evaporator. The yield of MEs was 3.2% (w/w) of the dried sample. 6-Hydroxydopamine (6-OHDA), 2,3-diaminonaphthalene, 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA), crystal violet, BCA assay kit, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and hematoxylin/eosin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) ELISA kits were purchased from Abcam (Cambridge, MA, USA). The Cell Death Detection kit was purchased from Roche Applied Science (Basel, Switzerland). The primary antibodies against IBA-1, p-ERK, p-JNK, p-p38, GAPDH, p-p65, p-Akt, tyrosine hydroxylase (TH), and α-synuclein as well as the horseradish peroxidase- (HRP-) conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 568 to goat IgG secondary antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA).