, 2001 and Wang et al., 1997). Deficiency of this vitamin is associated high throughput screening with impaired function of this cell type, including the reduction of its antimicrobial activity (Goldschmidt, 1991) and decreased spontaneous apoptosis (Vissers and Wilkie, 2007). Because both antioxidants are present in specific microenvironment in cells compartments, we believe that a combination of astaxanthin with vitamin C can improve the antioxidant effect of both. The purpose of the present study was to find out whether co-treatment of human neutrophils with high glucose (20 mM) and MGO can

alter the biochemical parameters of these immune cells. High glucose was used as a physiological intracellular source of MGO as previously described (Dhar et al., 2008). We also examined if astaxanthin associated with vitamin C can improve those biochemical

parameters. In addition, we evaluated the mechanism underlying this modulation. Methylglyoxal, D-glucose, astaxanthin, dihydroethidium, vitamin C, propidium iodide and most of the other chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA), find more except RPMI-1640 culture medium, lucigenin and pluronic acid, and acetoxymethylester (Fura-2AM), which came from Invitrogen (CA, USA). Common reagents for buffers (e.g. PBS) and regular laboratory solutions were obtained from Labsynth (Diadema, SP, Brazil). The Ethical Committee of the Universidade Cruzeiro do Sul approved the experimental procedure of this study. Around 30 healthy adult women and men (mean age 21.0 ± 4.0) were included in the present study. The subjects recruited did not present any systemic or topical therapeutic regimen, a smoking history, alcohol habits, obesity or any other systemic diseases at Oxymatrine least for the last 2 months (based on an anamnesis protocol). Neutrophils were obtained through the collection of human

peripheral blood by venipuncture procedure in vacuum/siliconized tubes containing 0.1 mM EDTA. Peripheral blood neutrophils were isolated under sterile conditions by using a density gradient present in the reagent Histopaque 1077 (Sigma–Aldrich), according to the manufacturer’s instruction. After obtained, neutrophils were counted in a Neubauer chamber using Trypan blue (1%). Neutrophils (1 × 106/mL) from each volunteer were cultured in 1 mL of RPMI-1640 medium supplemented with 10% fetal bovine serum, 20 mM Hepes, 2 mM glutamine, and antibiotics (streptomycin 100 units/mL and penicillin 200 units/mL) or ressuspended in Tyrode’s solution (137 mM NaCl, 2.68 mM KCl, 0.49 mM MgCl2, 12 mM NaHCO3, 0.36 mM NaH2PO4, 5.6 mM d-glucose, and 5 mM acid HEPES, pH 7.4) for acute assays. Before starting our experiments we evaluated the toxicity of increasing concentrations of MGO on neutrophils. For this purpose, cells (2.5 × 105) were treated for 18 h with MGO in concentrations ranging from 1 to 500 μM.