|
| Status |
Public on Mar 01, 2011 |
| Title |
healthy CD4 cells - after stimulation - proband35 |
| Sample type |
RNA |
| |
|
| Source name |
CD4 cells - after stimulation - proband35 - rep1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: peripheral blood CD4 cells
disease state: healthy
age in years: 64
used in fl/emzl vs healthy comparison (1-yes): 1
used in cll vs healthy comparison (1-yes): 0
used in mgus vs healthy comparison (1-yes): 0
|
| Treatment protocol |
For gene expression profiling and activation phenotyping, 10e5 purified CD4+ T cells were stimulated (10e6 cells/ml) on CD3-coated plates (BD Biosciences) with 3 µg/ml anti-CD28 (BD Biosciences) in AIM-V medium (Invitrogen) under serum-free conditions. After 40 h, cells were harvested and preserved in RNAlater (Ambion).
|
| Growth protocol |
CD4+ T cells were obtained from heparinized blood by Ficoll-Hypaque density gradient centrifugation and enriched to >95% purity (CD4+ T-cell isolation kit; Miltenyi Biotech) as verified by flow cytometry. In the case of CLL samples, B cells were depleted with microbead-conjugated CD19 antibodies (Miltenyi) prior to T cell purification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cryopreserved cell lysates (RNeasy Mini kit, QIAGEN), precipitated with ethanol, and reconstituted at 0.5 µg/µl. RNA quality was confirmed by a median RNA integrity number of 10 (range: 9.3-10) (Agilent 2100 Bioanalyzer; Agilent).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 5 μg total RNA with the Affymetrix “One-Cycle kit” (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 μg of cRNA were hybridized for 16 hr at 45C on U133 Plus 2.0 Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000 (Affymetrix)
|
| Description |
peripheral blood CD4 cells, purified (>95% purity), after 40h of in vitro stimulation (10e5 cells/well, 100μl/well) on CD3-coated plates (BD Biosciences Biocoat) with 3 µg/ml anti-CD28 (BD Biosciences) in AIM-V medium (Invitrogen) under serum-free conditions
Gene expression data from peripheral blood CD4 cells after in vitro stimulation with anti-CD3 and anti-CD28 for 40 h
|
| Data processing |
Microarray data were analyzed with Expressionist v5.3.3 software (Genedata). Gene expression values for subsequent analyses were calculated from raw data (.CEL files) with GC-RMA condensation after quantile normalization. Groups of differentially regulated genes cells were determined with an unpaired t-test including estimation of false-positive results by Benjamini-Hochberg Q values. The samples used in each group of patients and control donors are denoted above in the columns H-I (column title: characteristics: used in xx vs healthy comparison). The gene expression profiles corresponding to external, non-expression data, like the production of IFNγ or IL-4 in FL/eMZL CD4 cells, were determined through a “profile distance search” as correlations of ranks. The statistical significance of overrepresentation of members of specific pathways among various groups of deregulated genes was tested with Fisher’s exact test after import of the KEGG pathway annotations included in the current Bioconductor package v2.5 (annotation package hgu133plus2.db v2.3.5) based on KEGG release 52 (September 2009), which includes 358 human pathways (www.bioconductor.org).
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| |
|
| Submission date |
Jul 28, 2010 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Petros Christopoulos |
| Organization name |
Uniklinik Freiburg
|
| Department |
Medizin I.
|
| Street address |
Hugstetter Str. 55
|
| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE23293 |
Definition and characterization of the systemic T cell dysregulation in untreated indolent B cell lymphoma and very early CLL |
|
| Relations |
| Reanalyzed by |
GSE86362 |
