|
| Status |
Public on Oct 01, 2007 |
| Title |
Circulating CD4+ T cells stimulated with ConA AGM_stim_1_AB |
| Sample type |
RNA |
| |
|
| Source name |
circulating CD4+ T cells stimulated with ConA
|
| Organism |
Chlorocebus sabaeus |
| Characteristics |
African Green Monkey cells (chlorocebus sabaeus from Senegal).
Cells : CD4+ cells isolated from Peripheral Blood Mononuclear Cells (PBMC) consists of >95% of CD4+ T lymphocytes (CD3+ CD4+ cells).
Stimulus: 48H with Concanavalin A (ConA)
|
| Treatment protocol |
PBMC were isolated by Ficoll-Hypaque (Pharmacia Biotech) density gradient centrifugation and activated with 10µg/ml of ConA.
|
| Growth protocol |
For activation, 20 × 10^6 cells were plated in RPMI-1640 with 5% fetal calf serum (FCS) with ConA. The cells were harvested after 48H. The CD4+ cell subset was further isolated using the MACS magnetic labeling system (Miltenyi Biotec, Cologne, Germany) according to instructions of the manufacturer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 3 to 10 x 10^6 cells by using the RNeasy® Mini extraction Kit (Qiagen, Courtaboeuf, France) following the manufacturer's instructions.
|
| Label |
Chemiluminescence / DIG-UTP
|
| Label protocol |
Digoxigenin-UTP labeled cRNA was generated and amplified from 0.8µg of total RNA using Applied Biosystems Chemiluminescent RT-IVT Labeling Kit v1.0 according to the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
15µg of the labeled cRNA were hybridized to the ABI Human Whole Genome Arrays v1.0 Array using the Applied Biosystems Chemiluminescence Detection Kit accordingly to the protocol.
|
| Scan protocol |
Array processing, chemiluminescence detection, image acquisition, and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following manufacturer’s protocol.
|
| Description |
A pool of total RNA was prepared from enriched blood CD4+ T lymphocytes isolated from 5 AGM. The cells were previously activated with ConA for 48H. A unique sample was obtained and then divided to generate 4 aliquots used to hybridize 2 technical replicates for each of the ABI and Affymetrix platforms.
|
| Data processing |
Images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software.
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| |
|
| Submission date |
Feb 06, 2007 |
| Last update date |
Aug 23, 2007 |
| Contact name |
Beatrice JACQUELIN |
| E-mail(s) |
beajac@pasteur.fr
|
| Organization name |
Institut Pasteur
|
| Department |
Virologie
|
| Lab |
Regulations des Infections Retrovirales
|
| Street address |
25 rue du Docteur Roux
|
| City |
Paris cedex 15 |
| ZIP/Postal code |
75724 |
| Country |
France |
| |
|
| Platform ID |
GPL1426 |
| Series (1) |
| GSE6982 |
Long Oligonucleotide Microarrays for African Green Monkey Gene Expression Profile Analysis |
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