2 μg/mL phytohemagglutinin (PHA) and supplemented with 10 per cent foetal calf serum (Gibco, Eggenstein, Germany). Cell viability was 95 percent by the trypan blue exclusion test as described by Ribeiro DA et al. (17). The plate cultures of anaerobic group were incubated in sealed jars under an anaerobic environment produced by the method of Marshall (18). The RPMI 1640 medium cultured PBMC in 12 well plates of aerobic control groups were incubated in 5 per cent CO2 at 37°C. Samples of negative control group were cultured without PHA stimulation. At 6 hr and 24 hr hypoxia interference, 1 μg/mL Brefeldin A were added into culture wells and incubated

for 4 hr. The culture contents were then transferred into tubes and selleckchem centrifuged. The culture supernatants were removed and stored at −80°C until assay, while the remaining PBMC were resuspended at a concentration of 1 × 106 cells/mL for FACS. Prepared PBMC (100 μL) were stained for surface markers with 20 μL APC antihuman CD4 for 20 min at 4°C, washed twice with the staining buffer and fixed with 100 μL fixation buffer for 30 min at 4°C. After being permeabilized

with 1 mL permeabilization buffer for 30 min at 4°C, cells were then stained with 20 μL FITC antihuman IL-17A or FITC STI571 order antihuman IFN-γ for 30 min at 4°C. Cells were washed twice in permeabilizing solution, suspended with 0.5 mL staining buffer, and finally measured on a FACS Aria Cell Sorter (BD Biosciences Pharmingen Inc., San Diego, CA, USA). Isotype-matched monoclonal antibody were

used as controls. Purified APC-CD4 and FITC-IL-17A (or IFN-γ) double-positive lymphocytes were used as positive controls. The levels of IL-1β, IFN-γ, IL-23 and IL-17A protein in collected supernatants were double evaluated with commercially available ELISA kits following the procedures suggested by the manufacturer. The concentration of chemokines was determined spectrophotometrically. The absorbance was read at 450 nm. The assay sensitivity click here was less than 1 pg/mL. For each data group, results are expressed as means ± standard error of the mean and n refers to the number of independent experiments. Statistical analysis was performed using Student’s t-test (SPSS ver. 13.01). For statistical analysis, each treatment was compared with its respective control, and differences were considered significant at *P < 0.05, **P < 0.01 and ***P < 0.001. All Th1/Th17 ratios were obtained by FACS method and typical results are shown in Figure 1. Both Th1 and Th17 ratios presented upregulation after ischemia stimulation in SCI group but not in other groups. Th17 ratios peaked at 6 hr (mean 10.9%, n= 10) and diminished at 24 hr (mean 7.