|
| Status |
Public on Apr 24, 2007 |
| Title |
DC_Stimulated_Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
primary dendritic cells (DCs)
|
| Organism |
Homo sapiens |
| Characteristics |
Buffy coats were obtained from the National Blood Centre. Circulating myeloid dendritic cells (DCs) were isolated from buffy coats by adherence and culture in IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). DC preparations analyzed were more than 98% pure.
|
| Biomaterial provider |
UK National Blood Centre
|
| Treatment protocol |
Circulating myeloid DCs were isolated from buffy coats by adherence and culture in IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Immature primary monocyte derived DCs were stimulated for 2hr using plate bound anti-DC-SIGN antibody. To investigate the molecular nature of DC-SIGN signalling in primary dendritic cells (DCs), an antibody was identified that induces signalling in a similar manner to that produced following exposure of DCs to HIV-1. This antibody was used to activate primary DCs for two hours and the global gene expression profile documented.
|
| Growth protocol |
DCs were grown in R10 with IL4 and GM-CSF. Media changes occured every 48-72hr
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy kits (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
2 microgram RNA together with appropriate dilutions of GeneChip eukaryotic poly-A RNA spike controls (Affymetrix # 900433) was converted to double-stranded cDNA with superscript II (Invitrogen #11917-010) using T7-(dT)24 primer (Affymetrix 900375). This was used in an in vitro transcription to generate biotin-labelled cRNA probes.
|
| |
|
| Hybridization protocol |
Fragmented labelled cRNA (15µg) was used in 300µl hybridization cocktail containing spiked controls (Affymetrix #900299). A 200µl of this hybridization cocktail was used on the chip and incubated at 45°C for 16 h in the hybridization oven, rotating at 60 rpm. Following hybridization, the arrays were processed using a Genechip Fluidics Station 400 according to recommended protocols (EukGE-WS2v5, Affymetrix) of double-staining and post-hybridisations washes.
|
| Scan protocol |
Fluorescent images were captured using gene Array Scanner 3000 and GCOS1.2 software (Affymetrix).
|
| Description |
Circulating monocyte derived DCs were isolated from buffy coats by adherence and culture in IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). DC preparations analyzed were more than 98% pure. At day four 10 million immature DCs were either left unstimulated or stimulated using plate bound anti-DC-SIGN antibody for 2 hr. Three replicates of non-stimulated or stimulated cells were taken and used to extract total RNA.
|
| Data processing |
We have used the genechip robust multi-array average expression measurements (GC-RMA) (37-39) as implemented in BioConductor R statistics (www.bioconductor.org). Cell Intensity files (CEL) were used to obtain expression values for all the 54,675 probe sets (transcripts) on each of these arrays.
|
| |
|
| Submission date |
Oct 20, 2006 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Dilair Baban |
| E-mail(s) |
dilair.baban@well.ox.ac.uk
|
| Phone |
+44(0)1865287521
|
| Organization name |
University of Oxford
|
| Department |
Wellcome Trust Centre Human Genetics
|
| Lab |
Genomics
|
| Street address |
Roosevelt Drive
|
| City |
Oxford |
| ZIP/Postal code |
OX3 7BN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE6090 |
DC-SIGN initiates an immature dendritic cell phenotype triggering Rho activation that is utilised by HIV-1 |
|
| Relations |
| Reanalyzed by |
GSE49910 |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE86362 |
| Reanalyzed by |
GSE119087 |
