Hamster anti mouse cd11c clone n418 pe cyanine7 conjugate

BMDC in vivo migration was performed as previously described [28 (link)] with the following modifications. BMDC were generated from C57BL/6 mice as described above. BMDC were then transduced at an MOI of 50 with Ad5-LMP1, Ad5-Gag negative control, or Ad5-Gag DC matured with Mimic. After 36 hours, DC were CFSE labeled and injected intradermally into the flank of C57BL/6 mice. After 48 hours, the inguinal lymph nodes were dissected and processed into single cell suspensions. Cells were stained with hamster anti-mouse CD11c clone N418 PE-Cyanine7 conjugate (eBioscience) and analyzed by flow cytometry. The total number of migrated CD11c+CFSE+ dendritic cells was calculated based upon volume.

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.