7.6.4 flow cytometry analysis software

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.

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.