Pma ionomycin golgi plug

On day 5–6 of BM-DC culture, cells were gently removed and stained with the following antibodies (or appropriate isotype controls): CD16/CD32 Fc block (BD Biosciences, San Jose, CA), CD11c-APC (BD Biosciences), CD11c-PE-Cy7 (BD Biosciences), CD11b-PE (BD Biosciences), I-Ad-FITC (BD Biosciences), CD86-APC (BioLegend, San Diego, CA) or CD80-PE (BD Biosciences). Mean Fluorescence Intensity (MFI) of the above markers was determined on CD11c⁺ gated cells. On day 7, Leukocyte Activation Cocktail (PMA/Ionomycin/Golgi-plug; BD Biosciences) was added to culture media for the final 4 hours of culture. Cells were gently removed and stained with CD16/CD32 Fc block followed by CD11c-PE-Cy7. Cells were permeabilized and fixed (eBioscience, San Diego, CA) and then stained with anti-IL-12-PE (BD Biosciences) or anti-IL-6-PE (BD Biosciences).

Freshly prepared PBMCs with or without pDC depletion were cultured for 5 days in a 24-well plate stimulated with anti-CD3 (5 μg/ml) (BD-PharMingen) and anti-CD28 antibodies (3 μg/ml) (BD-PharMingen) in the presence or absence of 10 ug/ml CpG (ODN2336) (Coley Pharmaceutical, Wellesley, MA). At day 5, the above culture was stimulated with T cell activation cocktails (PMA+ionomycin+GolgiPlug) (BD-PharMingen) for 4 h, the cells were stained with anti-CD4-PE or –APC, then underwent intracellular staining for IFN-γ following instructions from the manufacturer (BD-PharMingen). IFN-γ-producing CD4⁺ T cells were analyzed by flow cytometry by gating CD4⁺ T cells. In some experiments, CD4⁺CD45RA⁺ T cells were purified through negative selection using StemCell Sep kit (StemCell Technology, Vancouver, Canada). Different numbers of pDCs (5000, 2500, 1250, 0) purified by BDCA-2 microbeads were incubated with autologous CD4⁺CD45RA⁺ T cells stimulated with CD3 and CD28 antibodies in the presence or absence of CpG as described above. IFN-γ in the supernatants was measured using Luminex assay kit (Millipore, Billerica, MA) and IFN-γ-producing cells were examined by intracellular cytokine staining.