5) for 30 min at 4 °C, followed by PBS wash (three times). Data were acquired using a FACSCalibur (BD Biosciences Pharmingen, San Jose, CA, USA) and analysed employing FlowJo 7.6.4 software (Tree Star Inc., Ashland, OR, USA). Single cell suspensions of peritoneal macrophages in each group (n = 10) were prepared as described above. Macrophages (2 x 105 cells/ml) were incubated with LPS (5 μg/ml) for 24 h. Then the culture supernatant was collected

for determination of cytokine. NO was determined by the Griess method as previously described ( Luna et al., 2012). Nitrite was used to assess NO and absorbance was measured at 550 nm by a microplate reader. The cytokines IL-1β, IL-6, IL-18 and TNF-α in culture supernatants were determined using ELISA kits (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions. The absorbance was measured at 450 nm by a microplate reader. The limit of detection PARP inhibitor for the cytokines shown was IL-1β

(12.5 pg/ml), IL-6 (7.8 pg/ml), IL-18 (15.6 pg/ml), and TNF-α (10.9 pg/ml). In addition, single cell suspensions of splenic cells in each group (n = 10) were prepared as described Etoposide manufacturer above. Splenic cells (2 x 105 cells/ml) were incubated either with ConA (5 μg/ml) for 48 h or phorbol-12-myristate-13-acetate (PMA; 50 ng/ml; Beyotime, Haimen, Jiangsu, China) and ionomycin (1 μg/ml; Beyotime, Haimen, Jiangsu, China) for 5 h. The culture supernatant was collected after the stimulation of ConA for the assessment of IL-10 and TNF-α, and after the stimulation of PMA and ionomycin Casein kinase 1 for the assessment of IL-4 and IFN-γ. The cytokines IL-4, IL-10, IFN-γ and TNF-α in culture supernatants were also determined using ELISA kits (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions.

The limit of detection for the cytokines shown was IL-4 (7.8 pg/ml), IL-10 (15.6 pg/ml), IFN-γ (9.4 pg/ml), and TNF-α (10.9 pg/ml). All data were analysed with SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Results are expressed as means ± standard deviation (SD). Statistical analysis for homogenous variance data was performed by one-way ANOVA and Tukey’s HSD test for multiple comparisons. Results were considered to be statistically significant at p < 0.05 (two-sided). During the entire exposure period in each group of animals, no behavioural or mental disorders were observed, the food and water consumption was normal, and the body hair was soft and smooth—all with no obvious clinical signs and symptoms. After 4 months of exposure, the body, thymus, and spleen weights of the mice in each group exhibited no significant differences (Table 1). The renal-function test results (including BUN and CR levels) for the mice in each group were within the normal range, with no significant difference being observed between the groups (Table 1).