C2: The bar figures of CD4+CD25+FoxP3+ Treg cells in various groups. early stage diseases and 10 advanced diseases) with ovarian carcinoma undergoing staging or debulking surgery were recruited. Stages I and II diseases were defined as early stages while stages III and IV were defined as advanced stages. The Institutional Review Board reviewed and approved the study protocol. The collection of cancerous tissue, ascites, and peripheral PBMCs were acquired after the patients signed informed consent. These ascites specimens were separated into supernatant and cellular components by centrifugation at 2000 rpm for 5 minutes. The supernatant was stored at ?20C and the cells were stored at ?135C until analysis. The disease stage of the ovarian cancer patients was based on the criteria of the International Federation of Gynecology and Obstetrics (FIGO) [20]. Tumor cell line The generation of WF-3 tumor cells was as previously described and maintained in RPMI-1640, supplemented with 10% (volume/volume) fetal bovine serum, 50 U/mL penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM non-essential amino acids, and 0.4 mg/mL G418 at 37C with 5% carbon dioxide [19]. Mice Six-to-eight week-old female C57BL/6J mice were bred in and purchased from the animal facility of the National Taiwan University Melagatran Hospital (Taipei, Taiwan). All animal procedures were conducted according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. Collection of ascites and tumor-associated cells (TACs) The WF-3 tumor cells (5104/mouse) were injected intra-peritoneally (6 mice per group) and the mice were sacrificed on days 14 and 49 post-injection. One ml phosphate-buffered saline (PBS) was injected into the peritoneal cavity and intra-peritoneal fluid was collected on day 14 after tumor injection while ascites was collected directly from mice 49 days after tumor injection. The ascites were separated into supernatant and cellular components as described earlier. The supernatant was stored at ?20C whereas cells defined as tumor-associated cells were stored at ?135C until analysis. Surface marker staining and flow cytometry of splenocytes and TACs For the animal part, the mice were first injected with WF-3 and sacrificed after tumor challenge as described earlier. The splenocytes were treated and obtained as described previously [21]. The splenocytes were then used directly or stored at ?135C until further experiments. The mice splenocytes and TACs were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD3 (Biolegend, San Diego, CA), phycoerythrin Melagatran (PE)-conjugated anti-mouse CD4 Melagatran (Biolegend), PE-conjugated anti-mouse CD8 (Biolegend), PE/Cy5-conjugated anti-mouse CD4 (eBioscience, San Diego, CA), PE-conjugated anti-mouse CD25 (eBioscience), PE-conjugated anti-mouse CD19 (eBioscience), PE-conjugated anti-mouse NK1.1(Biolegend), or PE-conjugated anti-mouse CD223 (eBioscience) for different experiments [22]. Flow cytometry assays and analyses were performed using a Becton Dickinson FACScan (Becton Dickinson, Franklin Lakes, NJ) with CELLQuest software. For the human part, the human TACs were stained with FITC-conjugated anti-human CD3 (Biolegend), FITC-conjugated anti-human Lin (Biolegend), PE-Cy5-conjugated anti-human CD4 (Biolegend), PE-Cy5-conjugated anti-human CD33 (Biolegend), PE-conjugated anti-human CD11b (Biolegend), PE-conjugated anti-human CD8 (BD Biosciences, San Diego, CA), or PE-conjugated anti-human CD25 (Biolegend) in different experiments, and analyzed by flow cytometry as described earlier. Immuno-histochemistry for CD4+FoxP3+ Treg cells Immuno-histochemistry studies of Treg cells in murine spleens were performed with some modifications [23]. Briefly, eight-micrometer cryostat sections were obtained from unfixed tissue embedded in optimal cutting heat (OCT) compound. After fixation with cold methanol (?20C) for 20 min, Rabbit Polyclonal to BTK (phospho-Tyr551) the sections were incubated with 5% fetal bovine serum (FBS) for 10 min. Subsequently, the sections were incubated at 4C overnight with the primary antibody, including rat anti-mouse CD4 antibody (Abcam, Cambridge, MA) and rabbit anti-mouse FoxP3 antibody (Abcam), and then washed three times in PBS for 15 min. After incubation with the primary antibody, the sections were then incubated at room heat for 1C2 hours with appropriate secondary antibodies like anti-rat secondary antibody-FITC (Abcam) and anti-rabbit secondary antibody-H&L-F(ab)2 fragment (Abcam) in PBS made up of 0.5% FBS, followed by counter-staining by Hoechst33342 (Sigma-Aldrich, St. Louis, MO). After several washings with PBS, the sections were cover-slipped using anti-fade mounting medium (Invitrogen, Carlsbad, CA) and analyzed by confocal microscopy (Leica TCS SP2, Heidelberg, Germany). Characterization of Tregs by flow cytometry To identify the Treg cells in murine splenocytes and TACs, splenocytes and TACs were first stained with PE/Cy5-conjugated anti-mouse CD4 (eBioscience) and PE-conjugated anti-mouse.