FACS–array profiling of helper T cell subtypes from human peripheral blood.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE59295 | GEO | 2014/11/13
SECONDARY ACCESSION(S): PRJNA254984
REPOSITORIES: GEO
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Project description:Gene expression analysis of Th1(CD4+/CXCR3+/CCR4-) and Th2 ((CD4+/CCR4+/CXCR3-) cells by FACS-array 12 Th1 and 12 Th2 samples
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Project description:We next sought to identify the transcriptional program that differentiates IL-9+Th2 cells from “conventional” Th2 cells. To this end, we selected representative Th1, Th17, Th2, and IL-9+Th2 clones (Figure 5A) and determined their transcriptome in the resting state and at different time points after activation using RNAseq. Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation. Human CD4+ T cells were isolated from PBMC by EasySep positive selection kit (Stemcell Technologies) according to manufacturer’s instruction. Positively selected CD4+ T cells were washed with PBS and stained for subsequent Th cell subset sorting. Memory Th cell subsets were sorted to over 90% purity according to their expression of chemokine receptors from CD45RA-CD25-CD8-CD3+ cells: Th1(CXCR3+CCR8-CCR6-CCR4-), Th2 (CXCR3-CCR8-CCR6-CCR4+), Th17 (CXCR3-CCR8-CCR6+CCR4+), Th9 (CXCR3-CCR8+CCR6-CCR4+). Single cell Th subset clones were directly sorted into 96well plate according to their expression of chemokine receptors (see above). Single cell clones were expanded and maintained by periodic restimulation with PHA (phytohaemaglutinine, 1 µg/ml, Sigma-Chemicals) and irradiated allogenic feeder cells (5x104/well) in culture medium. T cells were polyclonally activated using beads coated with antibodies against CD3, CD2, and CD28 (T cell/bead = 2:1, human T cell activation/expansion Kit, Miltenyi). Cell cultures were sampled before activation (time 0h) and 2, 4, 6, 9, 12, 24, and 48 hours after activation.
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