Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Real-time quantitative PCR analysis of human dendritic cells

ABSTRACT: Human CD14 positive monocytes were purified from healthy volunteers' blood and were differentiated to immature dendritic cells in vitro by culturing for five days in the presence of interleukin-4 (IL-4 100 ng/ml) and GM-CSF (75 ng/ml). Immature dendritic cells were activated three different ways for 24 hours: 1. Double-stranded DNA poly(dA:dT) (2.5 μg/ml) complexed with LyoVec transfection reagent. 2. LPS (500 ng/ml) 3. Inflammatory cocktail containing 10 ng/ml TNF, 5 ng/ml IL-1β, 20 ng/ml IL-6, 75 ng/ml GM-CSF and 1 μg/ml PGE2. We used SA Biosciences Antigen Presenting and Toll-like Receptor Pathway PCR Arrays to quantitate gene expression of immunologically relevant genes from the immature and the differently activated cells. Monocytes from three donors were used and treated separately as indicated in the summary. Equal amount total RNA from each donor was pooled prior to gene expression analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Attila Szanto

PROVIDER: E-GEOD-29131 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Cytosolic DNA-activated human dendritic cells are potent activators of the adaptive immune response.

Kis-Toth Katalin K Szanto Attila A Thai To-Ha TH Tsokos George C GC

Journal of immunology (Baltimore, Md. : 1950) 20110627 3

Recent studies in cell lines and genetically engineered mice have demonstrated that cytosolic dsDNA could activate dendritic cells (DCs) to become effector APCs. Recognition of DNA might be a major factor in antimicrobial immune responses against cytosolic pathogens and also in human autoimmune diseases such as systemic lupus erythematosus. However, the role of cytosolic dsDNA in human DC activation and its effects on effector T and B cells are still elusive. In this study, we demonstrate that i ...[more]

PMID: 21709148

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Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. NaÃ¯ve CD4+ T cells were isolated from buffy coat of four healthy donors using a naÃ¯ve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). NaÃ¯ve CD4+ T cells were stimulated with plate-bound anti-CD3 (500 ng/mL), soluble anti-CD28 (500 ng/mL), in the presence of IL-12 (5 ng/mL), IL-2 (10 ng/mL) and antiâ??IL-4 (5 Âµg/mL) for Th1, IL-4 (10 ng/mL), IL-2 (10 ng/mL) and anti-IL-12 (5 Âµg/mL) and antiâ??IFN-g (5 Âµg/mL) for Th2. Cells were cultured for six days in Iscoveâ??s modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 ÂµM Î²â??mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 ug/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were cultured for 6 days and then re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of corresponding polarizing cytokines and antibodies for another 2 days (Zhang et al. 2013). RNA was extracted using a miRneasy Mini Kit (Qiagen). The RNA concentrations were analysed with NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For the gene expression microarray analysis, The cRNA was prepared using a Low Input QuickAmp Labeling Kit. For Th1 and Th2 cells the gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8x60K microarray kit, according to the manufactureâ??s instruction (Agilent Technologies).

Dataset Information

Real-time quantitative PCR analysis of human dendritic cells

Publications

Cytosolic DNA-activated human dendritic cells are potent activators of the adaptive immune response.

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