Hla dr clone tu36

Monocyte derived DC were generated by standard methods [38 (link)] from PBMC obtained from anonymous donors (Continental Blood Services Inc, Miami) and plated on 6 well plates. DC were then transduced with Ad5-ΔLMP1-MAVS or Ad5-Gag 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. As a positive control, DC were matured with cytokine mix Mimic (5 ng/ml TNF-α (Peprotech), 5ng/ml IL-1b (Peprotech), 750ng/ml IL-6 (Peprotech), and 1ug/ml PGE2 (Sigma)). Cells were incubated for 36 hours at 37°C, harvested, and stained with the following antibodies: CD86 clone 2331 (FUN-1), CD80 clone L307.4, HLA-DR clone TU36, CD83 clone HB15e, CD40 clone 5C3, CD197 (CCR7) clone 3D12, and CD11c clone 3.9) (BD Bioscience). 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.

MDDC were matured and then stained for markers of maturation and activation (anti-human CD14 clone M5E2, CD86 clone 2331 (FUN-1), CD80 clone L307.4, HLA-DR clone TU36, CD83 clone HB15e, CD40 clone 5C3, CD197 clone 3D12, and CD11c clone 3.9) (BD Bioscience).