In the literature, the physiological concentration of MGO in plasma is about 5 μM, but levels can be 5–6 times higher in patients with diabetes types 1 and 2 (Dutra et al., 2005). Based on those data, the concentration of MGO selected to be used in the present study was 30 μM MGO (nontoxic, data not shown) in Tyrode’s solution. Glucose concentration was used at 20 mM, also confirmed as a nontoxic concentration

(Trypan blue exclusion, data not shown). Astaxanthin at 2 μM was solubilized in DMSO, whereas vitamin C at 100 μM was solubilized in Tyrode’s solution. The following experimental groups were created: control (without treatment), AV (astaxanthin + vitamin C), GM (glucose + methylglyoxal) and AVGM (astaxanthin + vitamin C + glucose + methylglyoxal). Cells were cultured at 5% CO2 for 18 h at 37 °C and then were collected, centrifuged and stored at −80 °C to assay glutathione MDV3100 research buy content and antioxidant enzyme activity. To measure cytokines release, cells were cultured for ABT-199 concentration 18 h and the supernatant was collected and stored under the same condition. ROS production and phagocytic capacity were assayed in neutrophils after acute treatment

of cells. To assess whether the concentration of MGO, glucose and both antioxidants astaxanthin and vitamin C selected for the experiments caused toxicity in neutrophils, we assayed cell viability by using flow cytometer analysis. Immediately after being obtained and at the end of the culture period (24 h), cells (5 × 105) were treated as previously described

and then used to test the membrane integrity. This assay was carried out in a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA) using propidium iodide (PI) (50 μg/mL) dissolved in phosphate buffered saline (0.137 M NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, pH 7.4). PI is a highly water-soluble fluorescent compound that cannot pass through intact membranes and is generally excluded from viable cells. When cells lose membrane integrity it passes through membrane and binds to DNA. Therefore, an increase in fluorescence to PI indicates a decrease in the proportion of viable cells. Fluorescence of PJ34 HCl PI was determined in FL2 channel (orange-red fluorescence-585/42 nm). The results were expressed as percentage of the control group. Neutrophils (5 × 105 cell/well) were treated and incubated for 60 min at 37 °C in 1 mL RPMI 1640 medium with opsonised zymosan particles. Zymosan particles (5 × 106/well) were opsonized by incubation in the presence of control serum for 60 min. Afterwards cells were harvested, citocentrifuged, stained and counted in an optical microscope. The score of phagocytosis was expressed by the number of cells that had one, two, three, four or more phagocyted zymosan particles (Sampaio et al., 2001). Production of HOCl by neutrophils was evaluated according to the method described by Dypbukt et al. (Dypbukt et al., 2005).