Cd86 clone 2331 fun 1

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.

For DC phenotype characterization, the following Abs were used: CD11c (clone 3.9 from Biolegend), HLA-DR (clone G46–6 from Biolegend), CD80 (clone L307.4 from Pharmingen), CD86 (clone 2331(FUN-1) from Pharmingen) and CD14 (clone M5E2 from Biolegend). Samples were analyzed using a FACSCanto flow cytometer (BD Biosciences).

DC and CIK cells cultured on day 1 and day 7 were collected. After washing with PBS and centrifugation, the DC cells were incubated with FITC-conjugated mouse anti-human CD14 (Clone M5E2, Cat. No. 561712, BD Pharmingen, US), CD83 (Clone HB15e, Cat. No. 560929, BD Pharmingen, US) and CD86 (Clone 2331 (FUN-1), Cat. No. 560958, BD Pharmingen, US) monoclonal antibodies and PE-labeled mouse anti-human HLA-DR monoclonal antibody (Clone G46–6, Cat. No. 560943, BD Pharmingen, US) for 20 min at room temperature. The CIK cells were incubated with FITC-conjugated mouse anti-human CD3 (Clone HIT3a, Cat. No. 561802, BD Pharmingen, US) and CD4 (Clone RPA-T4, Cat. No. 561005, BD Pharmingen, US) monoclonal antibodies and PE-labeled mouse anti-human CD8 (Clone RPA-T8, Cat. No. 561949 BD Pharmingen, US) and CD56 (Clone B159, Cat. No. 561903, BD Pharmingen, US) monoclonal antibodies for 20 min at room temperature. Then the DC and CIK cells were washed with PBS twice. Flow cytometry was used to determine the phenotypes of DC and CIK cells.