Cd83 clone michel 17

BMDC were generated from C57BL/6 mice as described above. 1x10⁶ DC were plated in each well of 6-well tissue culture-treated plates in a volume of 800ul. DC were transduced with Ad5-LMP1 or Ad5-GFP control (MOI = 50). DC were incubated with virus at 4°C for 1 h, followed by 3 h at 37°C. Complete media was then added to 2ml with a final concentration of 1ug/ml doxycyline. As a positive control, cytokine mix Mimic (5 ng/ml TNF-α (Peprotech), 5ng/ml IL-1b (Peprotech), 750ng/ml IL-6 (Peprotech), and 1ug/ml PGE2 (Sigma)) was used to mature DC. Cells were incubated for 36 hours at 37°C, harvested, and stained with the following antibodies: anti-mouse CD80 clone 16-10A1, CD86 clone GL1, CD40 clone 1C10, CD83 clone Michel-17, MHC Class II (I-A/I-E) clone M5-114.15.2, and CCR7 clone 4B12 (all from eBioscience). Tubes were also stained with hamster anti-mouse CD11c clone N418 PE-Cyanine7 conjugate (eBioscience) to allow for gating on CD11c+ DC. After flow cytometry analysis, the mean fluorescence intensity (MFI) for each antibody was calculated for CD11c+ dendritic cells under each experimental condition. FlowJo 7.6.4 flow cytometry analysis software (FlowJo, Ashland, OR) was used for analysis. Three independent wells were analyzed for each condition.

Dendritic cells were obtained from mouse bone marrow and cultivated in RPMI-1640 with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/mL of interleukine-4 (IL-4). On day 7, GC (50 μg/mL) was added to the culture medium. TNFα at a concentration of 20 ng/mL was used as a positive control. Three days after incubation, the DC cell surface molecules and cytokine production were determined by flow cytometry (Cytomix FC-500, Beckman Coulter, USA with the CXP software). The cell population gate was determined by forward and side scattering and cell size; 10,000 cells per gate were recorded. The Abs that was used included: FITC or PE-conjugated Abs against CD34 (clone RAM34, FITC), CD11c (clone HL3, FITC), CD83 (clone Michel-17, FITC), CD86 (clone PO3.1, PE), CD80 (clone 16-10A1, FITC), MHCII (I-EK, PE) (clone 14-4-45); all were obtained from eBioscience (eBioscience Inc., San Diego, CA, USA).