|
| Status |
Public on Feb 08, 2007 |
| Title |
Dendritic cells generated from blood of donor 2 |
| Sample type |
RNA |
| |
|
| Source name |
Immature dendritic cells cultivated in medium for 6h
|
| Organism |
Homo sapiens |
| Characteristics |
Buffy Coat blood of healthy donors
|
| Biomaterial provider |
DRK-Blutspendedienst, Frankfurt, Germany
|
| Treatment protocol |
Peripheral blood mononuclear cells (PBMCs) were separated from 50 ml Buffy Coat blood of healthy donors (DRK-Blutspendedienst, Frankfurt, Germany) by Ficoll-Hypaque density gradient centrifugation (Biochrom AG, Berlin, Germany). Human monocytes were isolated from PBMCs by magnetic-associated cell sorting (MACS) using paramagnetic microbeads conjugated to monoclonal anti-human CD14 antibodies (Miltenyi, Bergisch Gladbach, Germany).
|
| Growth protocol |
Differentiation of monocytes into immature DCs(iDCs) was achieved by cultivating cells in RPMI 1640 medium (Invitrogen, Karlsruhe, Germany) supplemented with 100 mg/ml Refobacin (Merck, Darmstadt, Germany), 10% fetal calf serum (FCS, Sigma-Aldrich, Taufkirchen, Germany), 100 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (Leukine, Novartis, Basel, Switzerland) and 20 ng/ml interleukin-4 (R&D Systems, Wiesbaden, Germany). Cultures were fed with fresh medium and cytokines every other day. After culturing for 6 days at 37°C and 5% CO2, iDCs were collected, washed with Hanks buffer and 1x 106 cells were resuspended in 1 ml complete medium. If iDCs were stimulated with fungi, 1 x 106 Aspergillus fumigatus germ tubes were added for 6h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy Mini kit and QiaShredder spin columns (Qiagen, Hilden, Germany) according to the manufacturer‘s protocol. RNA was eluated with 35 µl RNase-free water and quality and concentration was determined with the 2100 Bioanalyzer (Agilent, Waldbronn, Germany).
|
| Label |
Biotin
|
| Label protocol |
cRNA was prepared and biotin-labeled by in vitro transcription from 2 µg of total RNA according to the manufacturers protocol without modification (One-Cycle Labeling Kit, Affymetrix, Santa Clara, USA)
|
| |
|
| Hybridization protocol |
Labeled RNA was fragmented and incubated for 16 h at 45°C with a HG-U133 Plus 2.0 human genome array (Affymetrix). The gene chips were automatically washed and stained with streptavidin-phycoerythrin on a fluidics station.
|
| Scan protocol |
The probe arrays were scanned by using a GeneChip Scanner 3000 (Affymetrix).
|
| Description |
no additional information
|
| Data processing |
For evaluation and analysis of array data, software packages from the Bioconductor project (www.bioconductor.org; Gentleman et al., 2005) were run under R (www.r-project.org). Data were normalized using the function expresso from the affy-package, using variance stabilization normalization (VSN; Huber et al., 2002) and perfect match values only.
|
| |
|
| Submission date |
Feb 06, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Juergen Loeffler |
| E-mail(s) |
loeffler_j@klinik.uni-wuerzburg.de
|
| Phone |
04993120136412
|
| Fax |
04993120136409
|
| Organization name |
Universitätsklinikum Würzburg
|
| Department |
Medizinische Klinik & Poliklinik II
|
| Lab |
Molekulare Infektionsimmunologie
|
| Street address |
Josef-Schneider-Str. 2
|
| City |
Würzburg |
| ZIP/Postal code |
97070 |
| Country |
Germany |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE6965 |
Gene expression profile of human dendritic cells after infection with A. fumigatus |
|
| Relations |
| Reanalyzed by |
GSE49910 |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE86362 |
| Reanalyzed by |
GSE119087 |
