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Title: Impact of gamma chain cytokines on the differentiation of recently antigen-activated CD8 T cells      
keywords:
Transcriptome or Gene expression
ID:
PRJNA251757
description:
Analysis of how different gamma chain family cytokines influence CD8 T cell differentiation. Overall design: Naïve CD8 T cells were isolated from the spleens OT-I Thy1.1 TCR Tg mice. Whole splenocytes from wild-type C57BL/6 mice were used as stimulator cells. Purified naïve wild-type OT-I (1×106/well) were stimulated with OVA peptide (SIINFEKL) pulsed (5 µg/ml) and irradiated (2,000 rads) syngeneic splenocytes (6×106/well) in 24-well plates. Forty-eight hours later, activated OT-I T cells were harvested and viable cells were enriched over a Ficoll-paque gradient and washed with cRPMI prior to being reseeded in cRPMI (5×105 cells/ml) and treated with the media supplemented with various γc cytokines (IL-2, IL-4, IL-7, IL-15 and IL-21). Experimental samples were treated with 100 ng/ml of their respective gamma chain cytokine and incubated at 37ºC for 24 hours. RNA was isolated using either the RNeasy Mini kit, or a combination of TRIzol reagent and the Direct-zol RNA miniprep kit, all following manufacturer protocols. Two biological replicates for mRNA analysis were prepped using the RNeasy kit. The third replicate was prepped using the Trizol/Direct-zol approach. The quality and quantity of RNA samples was further analyzed on the Bioanalyzer. Labeled target cDNA was prepared from total RNA samples using the Ambion MessageAmp Premier protocol (3’IVT assay). Each sample target was hybridized to a Mouse 430 2.0 GeneChip array. Image processing and expression analysis were performed using Affymetrix GeneChip Command Console (AGCC) v. 3.1.1 and Affymetrix Expression Console v.1.1 software, respectively. Data from all biological replicates and conditions was imported into the Affymetrix Expression Console and normalized (RMA). RNA processing and microarray hybridization were performed by the Oregon Health & Science University Gene Microarray Shared Resource core facility in Portland, Oregon.
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA251757
authentication:
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authorization:
none
name:
Mus musculus
ncbiID:
ncbitax:10090
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject