Figure 4. Cytokine release from dog PBDC cultures. Peripheral blood cultures were collected after 7 days in culture with recombinant canine IL-4 and GM-CSF, or Ad.hFlt3L conditioned medium and incubated for additional 24 h without added cytokines in the absence (mock) or presence of CpG2216 or LPS. Release of IL-6 (A), TNF-a (B) and IFN-c (C) was determined by ELISA. *p,0.05 vs mock (Two-way ANOVA followed by Tukey’s Multiple Comparison test). Scatter plots show values from individual dog blood cultures (n = 4–12). doi:10.1371/journal.pone.0011074.g004 Human Flt3L Expands Dog DCs in 6-well plates (1.26107 cells/well, FALCONH, Cat # 353046) for 2 h at 37uC in 2 ml supplemented RPMI. After 2 h, about 30% of the cells had already attached. Non-adherent leucocytes were removed by repeated washes with phosphate buffered saline (PBS). Adherent PBMCs were then cultured with 2 ml culture medium containing hsFlt3L conditioned medium (final concentration of hsFlt3L: 10 ng/ml). As positive controls, adherent PBMCs were cultured in RPMI containing 50 ng/ml recombinant canine IL-4 (R&D Systems, Cat # 754-CL) plus 33 ng/ml recombinant canine GM-CSF (R&D Systems, Cat # 1546-GM). Half of the culture medium was replaced with fresh medium containing cytokines every 2,3 days. Non-adherent cells were harvested 7–10 days later and analyzed by flow cytometry or seeded in 24-well plates (,0.5–16106/well in 0.2 ml) to induce DC maturation. These cells were incubated for additional 24h without cytokines and in the presence of 5 mg/ml LPS (InvivoGen, Cat # tlrl-eklps) or 50 mg/ml ODN CpG2007 (Cell Sciences Hycult biotechnology, cat # HC4038) or ODN CpG 2216 (Alexis Biochemicals cat #TALX-746-005) to induce maturation of DCs. Supernatant was collected for detection of Th1 cytokine release and cells were harvested for flow cytometric analysis, phagocytosis assays or allogeneic MLR. Flow cytometry Flow cytometric analysis was performed on peripheral blood- derived dendritic cells (PBDCs) collected from cultures expanded in the presence of hsFlt3L or canine IL-4/GM-CSF, or on PBDCs incubated for additional 24 h in the absence of cytokines and with LPS or CpG. 75,000–1,000,000 cells were used for staining, depending on the yield of the culture. Cells were stained for 30 min with 50 ml RPE-conjugated mouse anti-human CD14 (1:5, ABD Serotec, cat # MCA1568PE) or RPE-conjugated mouse IgG2a (ABD Serotec, cat # MCA929PE) as isotype control. Also, cells were incubated with 50ml mouse anti-canine CD11c, MHCII, CD80 and CD18 (8 mg/ml, generated in the Department of Veterinary, Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA) or mouse IgG1 (eBiosciences, cat # 14-4714-82) as isotype control. Cells were washed and incubated with 50 ml FITC-conjugated goat anti-mouse 488 (1:400, Molecular Probes, cat # A-21121) for additional 30 min. Finally, cells were washed and resuspended in PBS with 1%FBS. Flow cytometric analysis was performed using a FACScan flow cytometer (Becton Dickinson). Data were analyzed using DAKO Summit v4.3 software. Phagocytosis assessment 24 h after treatment with LPS or CpG in the absence of cytokines (see above), PBDCs were seeded in 24-well plate and mixed with 1.0 mm yellow-green fluorescent FluoSpheres (30 beads per cell, Invitrogen, Molecular Probes, Cat # F8823). After 4 h at 37uC or 4uC as control, cells were harvested, washed and fixed with 4% PFA. Cells were then stained with 8 mg/ml mouse anti-canine CD11c and MHCII or mouse IgG1 isotype control (all described above). Cells were washed and incubated with goat anti-mouse Alexa 594 (1:400, Molecular Probes, cat # A11032). Nuclei were stained with DAPI (5 mg/ml, Molecular Probes) and mounted with ProLong Antifade (Molecular Probes). Confocal micrographs were obtained using a Leica confocal microscope TCS SP2 with AOBS equipped with a 405-nm violet-diode UV laser, 488-nm argon laser, and 594- and 633-nm helium- neon lasers; using a HCX PL APO 63 3 1.4 numerical aperture oil objective (Leica Microsystems Heidelberg, Mannheim, Germany). PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11074