Sun et al. Journal of Experimental & Clinical Cancer Research 2011, 30:82 http://www.jeccr.com/content/30/1/82 Page 3 of 10 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72° C, with a final extension cycle of 72°C for 10 min. PCR products were analyzed by separation on 2% agarose gels. Quantitative real-time RT-PCR detection of Foxp3 Foxp3 gene expressions in T cells from different co-cul- tures were also assessed by quantitative real-time RT- PCR using b-actin mRNA as an internal control. Foxp3 primers, sense 5’-CCCACTTACAGGCACTCCTC-3’; antisense 5’-CTTCTCCTTCTCCAGCACCA-3’; b-actin, sense 5’-TGGCACCCAGCACAATGAA-3’; antisense 5’- CTAAGTCATAGTCCGCCTAGAAGCA-3’. PCR ampli- fications were performed in a 20 μl volume with each reaction containing 2 μl of 10× buffer, 0.4 μl (10 mmol/ l) dNTP mixture, 1 μl (10 μmol/l) of each primer, 2 μl cDNA, 1 μl (20×) SYBR Green I, 3.2 μl (25 mmol/l) MgCl2, 1 U Taq DNA polymerase, 2.0 μl (1 mg/ml) BSA and 6.4 μl ddH2O. The thermal cycling conditions used were 95°C for 5 min, 94°C for 20 s, 60°C for 30 s, 72°C for 20 s, 80°C for 1 s; this was repeated for 40 cycles. All samples were measured in duplicate, and the average value was quantitated. To correct for sample-to- sample variation, an endogenous control, b-actin, was amplified with the target and served as an internal refer- ence to normalize the data. The expression levels of Foxp3 relative to that ofb-actin were calculated by using the 2-ddCt method. Western blot analysis Total cellular extracts for Western blot analysis were obtained by lysis of 1 × 107 positively cloned CHO cells in lysis buffer (Pierce Biochemical, Rockford, IL), and the protein concentration was quantitated using the Micro BCA protein assay kit (Pierce). The extracts were heat denatured for 10 min in a 100°C water bath. Aliquots of cell lysates containing 50 μg of proteins were separated on a 12% SDS-polyacrylamide gel and transferred to PVDF membranes (Pall Corporation, Ann Arbor, MI). The filters were blocked with TBST buffer containing 2% BSA and incubated with an IDO monoclonal antibody (Chemicon International, Teme- cula, CA, 1:1000) overnight. Horseradish peroxidase- linked anti-mouse IgG (Chemicon, 1:5000) was then added, followed by immersion in SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnol- ogy, Rockford, IL) for visualization of bands. The intensity of each band was recorded using the Chemi- Doc XRS imaging system and was analyzed using Quantity One software (Bio-rad Laboratories, Milan, Italy). For detection of Foxp3 in co-cultures of IDO+ and CD3+ T cells (using mouse monoclonal antibody to Foxp3 [Clone PCH101, 1:1000 dilution; eBioscience]), inadherent cells were obtained 7 days after co-culture of CHO+ and CD3+ T cells, and the analysis was performed as described above. IDO activity assay IDO expressing or untransfected (control) CHO cells (1 × 107) were incubated in RPMI 1640 with 10% FBS (Gibco). The supernatants of cell culture were harvested 72 h after incubation, and 2 mls were added to 0.1 g sulfosalicylic acid, followed by centrifugation at 4°C for 30 min. The concentrations of the enzymatic products were measured using the Hitachi amino acid L-8800- automatic analyzer (Hitachi, Tokyo, Japan). Enzyme activity was expressed as the product content per hour per milligram of protein. Co-culture of IDO+ CHO cells and CD3+T cells Mononuclear cells were isolated from the peripheral blood of breast cancer patients using the CS-3000 Plus Blood Cell Separator (Baxter, Munich, Germany) according to the operator’s manual. CD3+T cells were isolated and purified using the RosetteSep Human CD3 Depletion Cocktail kit (StemCell Technologies Inc., Vancouver, BC, Canada) according to the manufac- turer’s instructions. Informed consent was obtained from all subjects, and the study was approved by the University Ethics Committee. CHO/EGFP cells or CHO cells with stable IDO expression (1 × 105) were seeded per well of a 24-well plate, and 2 × 106 purified CD3+T cells and 200 U/ml human recombinant IL-2 (R&D Sys- tems) were added. The cells were incubated in RPMI 1640 medium with 10% FBS at 37°C in a 5% CO2 incu- bator. The medium was changed every 2-3 days for 7 days. We added 1-MT, the specific inhibitor of IDO at concentration of 1 mM in the co-culture system com- posing of CHO/IDO cells and CD3+T cells to elucidate the regulatory effect of IDO both in promoting apopto- sis and increasing Tregs. Flow cytometry assay Co-cultured cells were harvested after 96 h for analysis of apoptosis. The apoptosis levels of T cells in the har- vested cells (1 × 106/ml), which were gated using PE- Cy5 labeled anti-CD3 monoclonal antibody, were assessed by FITC labeled Annexin V and PI (BD Phar- mingen, San Diego, CA) staining. As a positive control for apoptosis, CD3+ T cell apoptosis was also assessed 96 h after incubation in medium supplemented with 200 U/ml IL-2. To detect the proportion of Tregs after 7 days of co-culture, cells were harvested and incubated with 10 μl anti-CD4-PE-Cy5, 10 μl anti-CD25-FITC and 3 μl anti-CD127-PE (BD Pharmingen) at 4°C for 30 min in the dark. A minimum of 1 × 104 cells were washed 2 times with PBS and resuspended in 2% paraformalde- hyde. Flow cytometric analysis was performed using a