Journal: Clinical and Experimental Immunology
Article Title: Early exposure of interferon-? inhibits signal transducer and activator of transcription-6 signalling and nuclear factor ?B activation in a short-term monocyte-derived dendritic cell culture promoting 'FAST' regulatory dendritic cells
doi: 10.1111/j.1365-2249.2011.04537.x
Figure Lengend Snippet: Interferon (IFN)-γ in combination with the FAST-dendritic cell (DC) protocol generates phenotypically tolerogenic DC. Monocytes were isolated from peripheral blood and cultured without (UT-DC) or with IFN-γ (IFN-γ-DC) in the presence of interleukin (IL)-4 and granulocyte–macrophage colony-stimulating factor (GM-CSF) for 24 h. DC were then either matured with tumour necrosis factor (TNF)-α (10 ng/ml) and prostaglandin E2 (PGE2) (1 µM) for another 24 h (a: plus TNF-α and PGE2, representative of six independent experiments) or were cultured for another 24 h in the absence (b: minus TNF-α and PGE2, representative of three independent experiments. Fluorescence activated cell sorter (FACS) analysis was used to determine phenotypic markers. The following co-stimulatory and linage markers were examined: CD83, CD80, CD86, immunoglobulin (Ig)-like transcript 4 (ILT4), CD11c, DC-SIGN, CD14, human leucocyte antigen D-related (HLA-DR). Line indicates the isotype control and solid histogram shows the tested monoclonal antibody. Negative gate was set to <0·5%.
Article Snippet: Antibodies The phenotypic profile of DC was defined using the following directly conjugated monoclonal antibodies (mAb): anti-CD83-fluorescein isothiocyanate (FITC) (HB15e), anti-CD86-FITC (FUN1), anti-CD80-FITC (L307·4), anti-DC-SIGN-FITC (DCN46), anti-human leucocyte antigen D-related (HLA-DR)-phycoerythrin-cyanin-5 (PE-Cy5) (G46-6) (BD Bioscience, San Jose, CA, USA) and rat anti-human immunoglobulin (Ig)-like transcript 4 (ILT4) (42D1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a primary mAb and FITC-conjugated anti-rat IgG was used for detection.
Techniques: Isolation, Cell Culture, Fluorescence, Control