|
| Status |
Public on Jan 09, 2009 |
| Title |
TregB3_T24 |
| Sample type |
RNA |
| |
|
| Source name |
CD4 Treg clone, activated T=24 hrs
|
| Organism |
Homo sapiens |
| Characteristics |
From melanoma patient EB81
|
| Treatment protocol |
Cells were seeded in wells coated with 1 microg/ml anti-CD3 (Orthoclone OKT3, Janssen-Cilag), in the presence of 1 microg/ml soluble anti-CD28 (BD Pharmingen)
|
| Growth protocol |
Culture medium was IMDM supplemented with 10% human serum, L-arginine, L-asparagine, L-glutamine and methyl-tryptophane (200 microM). Treg and Th clones from hemochromatosis patient LB2050 were derived from flow cytometry-sorted CD4+CD25+ or CD4+CD25- PBMCs. Cells were seeded in limiting dilution conditions and stimulated weekly or bi-weekly with 1 microg/ml anti-CD3 antibody (Orthoclone OKT3, Janssen-Cilag), in the presence of 130 IU/ml of recombinant human IL-2 (Proleukin, Chiron), and irradiated feeder cells (5E05/ml allogeneic PBMCs and 5E05/ml cells of an allogeneic EBV-transformed B cell line). For long term maintenance of Treg clones, culture conditions were modified to increase the concentrations of IL-2 (1000 IU/ml) and irradiated allogeneic PBMCs (1E06/ml), and to include IL-4 (0,5 ng/ml) and IL-7 (10 ng/ml). Treg and Th clones from vaccinated melanoma patient EB81 were isolated in similar conditions from the TILs of a lymph node metastasis, except for anti-tumor clone Th B1, which was isolated from a Mixed Lymphocyte-Tumor Culture as previously described.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Tripure isolation reagent (Roche) according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 2-5 ug total RNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 20 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix GeneChip Human Genome U133A 2.0 Array. GeneChips were washed and stained in the Affymetrix genechip Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G3000A.
|
| Description |
Gene expression data from Treg clone, activated T=24 hrs
Experiment_number: I: Treg versus Th
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.
|
| |
|
| Submission date |
Jan 08, 2009 |
| Last update date |
Jan 08, 2009 |
| Contact name |
Sophie Lucas |
| E-mail(s) |
Sophie.Lucas@uclouvain.be
|
| Phone |
+32 2 764 7558
|
| Organization name |
de Duve Institute
|
| Street address |
avenue Hippocrate 74
|
| City |
Brussels |
| ZIP/Postal code |
1200 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE14330 |
Comparison of stable human Treg and Th clones by transcriptional profiling - experiment I |
|
