Erythrocytes were lysed by adding ammonium chloride solution (0.13 M) to the samples, and leukocytes were recovered after washing with PBS. Fluorescent dye DCFH-DA (340 μM; diluted in PBS) was added to 2 × 105 cells in a final volume of 1.1 ml. Cells were maintained at 37 °C for 30 min and rinsed
with EDTA (3 mM; 2 ml) to remove the excess dye. Cells were resuspended with PBS. The cells were analyzed in a FACS Calibur flow cytometer (Becton & Dickinson, San Jose, CA, USA). Data from 10,000 events were obtained and only the morphologically viable leukocytes were considered for analysis. Results are presented as arbitrary units of fluorescence. The effects of in vivo exposure to HQ on cell cycle and DNA fragmentation were studied using flow cytometry as previous described by Liu et al. (2005). Blood was collected, using heparin as anti-coagulant, from the GSK2656157 abdominal aorta of vehicle- or HQ-exposed mice, and erythrocytes Ribociclib datasheet were lysed by the addition of ammonium chloride solution (0.13 M). Leukocytes were recovered after washing with Hank’s balanced salt solution (HBSS). Afterward, RNAse A (20 μl; 15 mg/ml) and lysis buffer (140 μl; 2% fetal bovine serum, 0.05% Triton X 100, 0.1% sodium citrate in PBS) containing propidium iodide (20 μg/ml) were added to the leukocytes (1 × 105 cells). The samples were maintained
at room temperature for 30 min and immediately analyzed in a FACS Calibur flow cytometer (Becton & Dickinson, San Jose, CA, USA). Data from 10,000 events were obtained. Results of DNA fragmentation are presented as mean of arbitrary fluorescence units and cell cycle as percentage of labelled cells in each phase. As a positive
control, leukocytes were previously incubated with 10% dimethyl sulfoxide. The means and standard error of the mean (s.e.m.) of all data presented here were compared by Student’s t-test or ANOVA. Tukey’s multiple comparisons test was used to determine the significance of differences between the values for the experimental conditions. The statistical software GraphPad Prism® was used for this purpose. P < 0.05 was considered significant. To see more determine the amount of HQ in the exposure chamber, extracts of the cellulose ester membrane filters exposed for 1 h to 25 ppm HQ were analyzed by HPLC. The data obtained showed that the amount of HQ in the filter was 1.59 μg ± 0.26 (n = 5), which gives a concentration of 0.20 mg/m3 ± 0.09 in the box (according to NIOSH, protocol 5004). This concentration is equivalent to 0.04 ppm HQ (http://www.cdc.gov/niosh/docs/2004-101/calc.htm) and it is 10× lower than the level allowed for human exposure during a course of 8 h/day (0.44 ppm, threshold limit value − time weighted average (TLV − TWA); NIOSH, 1994).