|
| Status |
Public on Feb 15, 2011 |
| Title |
3_CD34+_12h_TCDD- |
| Sample type |
RNA |
| |
|
| Source name |
CD34+ cells from untreated normal donor
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: hematopoietic progenitor CD34+ cells
treatment: untreated
normal donor 3
|
| Treatment protocol |
The hematopoietic progenitor CD34+ cells were separated from the leukapheresis of normal donors stimulated with G-CSF using the immunonomagnetic MACS Miltenyi method. The cells harvested by apheresis were layered on a Ficoll-Paque gradient (specific gravity 1.077 g/ml; Nycomed Pharma AS, Oslo, Norway) in order to separate the low density mononuclear cells (LD-MNCs), and the samples were washed twice in Hanks' balanced salt solution (HBSS). The LD-MNCs were incubated with the QBEND10 monoclonal antibody directed against the CD34 antigen for 15 minutes at 4°C, and were then washed and incubated with immunomagnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) against QBEND10 for a further 15 minutes at 4°C. For the flow cytometry analysis, CD34-phycoerythrin (CD34-PE) conjugated antibody (HPCA-2, Becton Dickinson, Mountain View, CA, USA) was added to the cells for 15 minutes at 4°C. At the end of the separation, the cells were counted and assessed for viability by means of trypan blue dye exclusion, and their purity was determined by means of flow cytometry. All of these steps were performed under aseptic conditions. The mean purity of the CD34+ cells after immunomagnetic separation was 96.0%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions (GibcoBRL). RNA was purified using the RNeasy® Mini Kit according to the manufacturer's instruction (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 3 micrograms of total RNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 micrograms of cRNA were hybridized for 16 hr 30 minutes at 45°C on GeneChip HG-U133A Array. The arrays were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
The HG-U133A arrays were scanned using the GeneChip® Scanner 3000 7G (Affymetrix Inc., Santa Clara, CA).
|
| Description |
Gene expression profiling data of highly purified CD34+ cells from untreated normal donor 3.
|
| Data processing |
The data were analyzed with GeneChip Operating Software (GCOS version 1.4) using Affymetrix default analysis settings and global scaling as normalization.
|
| |
|
| Submission date |
Sep 17, 2010 |
| Last update date |
Feb 15, 2011 |
| Contact name |
Luca Agnelli |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
+390223903581
|
| Organization name |
IRCCS Istituto Nazionale dei Tumori
|
| Department |
Department of Advanced Diagnostics
|
| Street address |
Venezian 1
|
| City |
MILAN |
| ZIP/Postal code |
20133 |
| Country |
Italy |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE24193 |
Dioxin exposure of human CD34+ hemopoietic cells induces gene expression modulation that recapitulates its in vivo clinical and biological effects |
|
