Briefly, cells were pelleted and incubated with anti-CD11c MAC beads (400μL/108cells) (Miltenyi Biotec, Auburn, CA, USA) in the presence of 0.5% FCS and 2mM EDTA in PBS at 4°C for 15min. Cells were washed,

resuspended, and purified using the autoMACS system (Miltenyi Biotec) following manufacturer’s instructions. The percentage of CD11c+ cells purified in this manner was above 94% as measured by FACS analysis. 2.3. DC Maturation To precondition DC for IFN-gamma studies, DC monolayers were incubated Inhibitors,research,lifescience,medical in complete media containing 10ng/mL IFN-gamma for 2 hours. Cells were then washed and stimulated with either 1μg/mL LPS (derived from Escherichia coli (0111:B4) Sigma, San Diego, USA), 20μg/mL zymosan A (from Saccharomyces cerevisiae, Inhibitors,research,lifescience,medical Sigma) or 10μg/mL CpG1668 (GeneWorks, Adelaide, Australia) for 16h at 37°C. This procedure was previously optimized using the DC2.4 cell line (data not shown). Cells (5 × 105) were washed and resuspended with FITC-conjugated anti-CD40 (FGK-45.5), anti-CD80 (16.10.A1), anti-CD86 (GL1), anti-MHC-class II (IAb) (M5/114.15.2), all constructed in house, or PE-conjugated anti-MHC-class

I (BD BioSciences), together with APC-conjugated anti-CD11c (BD Biosciences) at 4°C for 30min. Cells Inhibitors,research,lifescience,medical were then analyzed for expression of surface maturation markers by gating on live CD11c+ cells. 2.4. T Cell Purification Splenocytes from C57BL/6 or OT-II mice were collected, washed, and incubated in red blood cell lysis buffer Inhibitors,research,lifescience,medical at room temperature for 5min. Cells were incubated with antibody mix which contained Selleck Alvocidib in-house produced rat anti-mouse Gr-1 (RB6-8C5), anti-CD11b (M1/70.15), anti-erythrocyte (TER-119), and anti-MHC-class II (M5/114.15.2) monoclonal antibodies at 4°C for 30min. To purify CD4+ and CD8+ T cells, rat anti-mouse CD8-alpha (YTS169.4)

and anti-CD4 (GK1.5) were included in the antibody mix, respectively. Labeled cells were depleted with 2 rounds of bead separation. In each round, cells were incubated with goat anti-rat Inhibitors,research,lifescience,medical Ig magnetic beads (8 beads/cell) (Qiagen, Melbourne, Australia) at 4°C for 25 min. Cells were washed and those that bound to the beads were removed below by magnets. The purity of T cells was at least 94%. 2.5. Antigen-Specific T Cell Proliferation Purified DCs were preconditioned with IFN-gamma (10ng/mL) for 2h and subsequently treated with endotoxin-depleted OVA (40μg/mL) and LPS (1μg/mL) or zymosan (20μg/mL) for 3h. To evaluate the capacity of treated DCs to stimulate OVA-specific helper T cells, titrated DCs (1–4 × 103) were seeded with 2 × 104 purified OT-II CD4+ T cells in quadruplicates in 96-well plates. Proliferation of T cells was monitored by the addition of 1μCi 3H-thymidine from day 1 to day 5. The radioactivity was measured in counts per minute (CPM). Peak proliferation of OT-II T cells on day 3 was compared. 2.6.