After 30 min incubation in the dark, cells were washed and analyzed by flow cytometry. Antigen presenting cells (SmyleDCs, SmartDCs or PBMCs) were irradiated with 30Gy, and CD3+ T cells isolated with immunobeads (Miltenyi Biotech) were used as responders. Different APC or PBMC ratios were co-cultured with 1 × 105 allogeneic CD3+ T cells (2, 5 and 20) in rounded-bottom 96-well plates in a total volume of

200 μL Cellgro medium. Triplicate wells were set up for each reaction and ratio. The reactions were incubated for 6 days at 37 °C. For the last 18 h of the culture, the supernatants from each reaction were collected for LY294002 price multiplex luminex bead kit. 1 μCi/well of [3H] Thymidine was added and [3H] Thymidine incorporation in the cells was measured on a β-scintillation counter. The stimulatory

capacity was determined with stimulation index (SI) = counts per minute (cpm) of stimulated T cells and stimulators/cpm of unstimulated T cells. To determine the production of cytokines by NK cells stimulated with iDCs, autologous NK cells were freshly isolated from PBMCs and co-incubated with 7 day SmyleDCs or SmartDCs at 1–5 ratio for 15–17 h. Staining of surface antigens on stimulated CD3−CD56+ NK cells was performed at 4 °C for 30 min. For selleck chemicals llc analysis of IFN-γ and TNF-α intracellular staining, cells were washed and fixed with 4% paraformaldehyde Non-specific serine/threonine protein kinase for 10 min. After fixation, cells were permeabilized with saponin buffer (PBS supplemented with 0.1% saponin and 10 mM HEPES) and stained with IFN-γ and TNF-α mAb. After 30 min incubation, cells were washed three times and the percentage of IFN-γ and TNF-α positive NK cells was determined by flow cytometry. SmyleDCs and SmartDCs kept in culture for 7 and 14 days were analyzed for their DC immunophenotype. Cell were harvested and washed once with PBS and blocked with mouse IgG (50 μg/mL) on ice for 15 min followed by staining with a combination of monoclonal antibodies; FITC-conjugated anti-human

CD209, APC-conjugated anti-human CD86, PE-conjugated anti-human CD80, PerCP-conjugated anti-human HLA-DR, PE-conjugated anti-human CD14 and PerCP-conjugated anti-human CD123 (Becton Dickinson) for 30 min in the dark. After washing off the unbound antibodies, cells were then resuspended in 1% paraformaldehyde for fixation and further analyzed with a FACS Calibur apparatus (Becton Dickinson), using CellQuest software. Total viable cells were gated and 20,000 cells in gate were acquired. 7-day conventional IL-4-DCs or IFN-α-DCs or iDCs (non-matured) or 5-day iDCs further incubated for 2 days with 200 IU/ml rhTNF-α, 5 ng/ml rhIL-1B, 10 ng/ml rhIL-6 and 1 mg/ml PGE2 (matured) were harvested and loaded with 10 μg/ml PepTivator CMV-pp65 overlapping peptide pool (Miltenyi Biotec). After 2 h, excess unloaded peptides were washed off.