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Real-time quantitative PCR analysis of human mononuclear cells from bone marrow.

ABSTRACT: Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE61819 | GEO | 2014/09/27

SECONDARY ACCESSION(S): PRJNA262388

REPOSITORIES: GEO

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Real-time quantitative PCR analysis of human mononuclear cells from bone marrow.

Project description:Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis Two condition experiment. Biological replicates: 3 control human healty subjects, 3 PMF patients

2014-09-27 | E-GEOD-61819 | biostudies-arrayexpress

Real-time quantitative PCR analysis of human mononuclear cells from bone marrow

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Real-time quantitative PCR analysis of mouse natural killer cells isolated from the bone marrow

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Real-time quantitative PCR analysis of mouse natural killer cells isolated from the bone marrow

Project description:A large gap in our understanding of infant immunity is why natural killer (NK) cell responses are deficient, which makes infants more prone to viral infection. Here we demonstrate that transforming growth factor-beta (TGF-beta) was responsible for NK cell immaturity during infancy. We found more fully mature NK cells in CD11cdnR mice, whose NK cells lack TGF-beta receptor (TGF-beta R) signaling. Ontogenic maturation of NK cells progressed faster in the absence of TGF-beta signaling, which results in the formation of a mature NK cell pool early in life. As a consequence, infant CD11cdnR mice efficiently controlled viral infections. These data thus demonstrate an unprecedented role for TGF-beta in ontogeny that can explain why NK cell responses are deficient early in life. Bone marrow cells were isolated from CD11cdnR (TG) and wild-type (WT) mice, and NK cells were sorted at different stages of development (stages D, E, and F) using BD Biosciences FACSAria. mNK cells from stages D, E, and F were obtained from three cell sorting analyses with samples pooled from n = 12 CD11cdnR and 25 WT mice. Equal amounts of total RNA from each stage was pooled prior to gene expression analysis. RNA was prepared using the RNeasy Micro kit (Qiagen), and cDNA was obtained using the standard protocol of reverse transcription. A customized StellARay cell cycle qPCR array (Lonza) was used. Quantitative gene expression analysis (quantitative PCR) was conducted on an ABI Prism 7900 instrument (Applied Biosystems).

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Real-time quantitative PCR analysis of human induced T regulatory cells (Tregs) for metabolic enzymes

Project description:Human naïve CD4 T cells were purified from healthy volunteers' blood and were differentiated into induced Tregs in vitro by culturing for 5 days in the presence of anti-CD3 and CD28 antibodies (2 µg/ml each), IL-2 (100 units/ml) and TGF-β1 (5 ng/ml) for 5 days, then in presence of anti-CD3 and CD28 antibodies for 2 days. Human iTregs were harvested on day 7 post differentiation, then treated with either vehicle or IL-21 (100 ng/ml) in serum-free RPMI media for 18 hours.

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