Transcriptomics

Dataset Information

5

PBMC LNA-array microRNA profiling


ABSTRACT: T cells were collected from four healthy donors using heparin anti-coagulant, diluted with equal amounts of PBS EDTA, then layered over Lymphoprep and spun to collect PBMCs. CD4+ T cells were purifed using the Miltenyi positive selection magnetic labeling kit. Cells were resuspended in RPMI (Invitrogen) with 10% FCS amd 50 U/ml IL-2 (Immunotools), and 3.5x105 plated out per well in 48 well plates, coated overnight with 2 µg/ml anti-CD3 and either anti-CD28 or anti-CD46. Purity was assessed by flow cytometry and was in all cases ≥ 95%. RNA was purified from a set of unactivated cells before plating, to represent the resting, 0 hr time point samples. For early activation samples, CD28 or CD46 co-stimulated cells were harvested 2 hrs after plating. Then, 36 hours after plating, the remaining CD46-stimulated cells were harvested and labeled for IL-10 or IFN-g secretion using the Miltenyi cytokine detection kits, according to manufacturer’s instructions. Cells were then sorted into separate cytokine-secreting populations (IFN-g+/IL-10–, IFN-g+/IL-10+, and IFN-g–/IL-10+) using a BD biosciences FACS machine.RNA from separate populations was labeled using the Hy3 microRNA (miRNA) Power labeling kit (Exiqon) and hybridised to an LNA (locked nucleic acid)-based miRCURY miRNA microarray slides (Exiqon, version 11) using a Maui hybridisation chamber (BioMicrosystems). Spot signals were acquired with an Agilent array scanner and GenePix Pro 4.1 was used for annotation (mirBase 14.0), signal processing and quantification using default background subtraction.

ORGANISM(S): Homo sapiens  

SUBMITTER: Jonathan Lou S Esguerra   Anna M Blom   Ben C King  

PROVIDER: E-MTAB-2658 | ArrayExpress | 2016-07-03

REPOSITORIES: ArrayExpress

Dataset's files

Source:
Action DRS
E-MTAB-2658.idf.txt Idf
E-MTAB-2658.idf.txt_original Idf
E-MTAB-2658.processed.1.zip Processed
E-MTAB-2658.sdrf.txt Txt
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Publications

CD46 Activation Regulates miR-150-Mediated Control of GLUT1 Expression and Cytokine Secretion in Human CD4+ T Cells.

King Ben C BC   Esguerra Jonathan L S JL   Golec Ewelina E   Eliasson Lena L   Kemper Claudia C   Blom Anna M AM  

Journal of immunology (Baltimore, Md. : 1950) 20160108 4


CD46 is a cell surface complement inhibitor widely expressed in human tissues, in contrast to mice, where expression is limited to the testes. In humans, it has been identified as an important T cell costimulatory receptor, and patients deficient in CD46 or its endogenous ligands are unable to mount effective Th1 T cell responses. Stimulation of human CD4(+) T cells with CD3 and CD46 also leads to the differentiation of a "switched" Th1 population, which shuts down IFN-γ secretion and upregulate  ...[more]

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