|
| Status |
Public on Dec 31, 2013 |
| Title |
CD8 T cells_untreated_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Naive T cells, Lymph node
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: B10D2
tissue: lymph node
cell type: Naive TCR transgenic T cells (CD44 negative) sorted by flow cytometry
|
| Treatment protocol |
For untreated samples, Naive CD8 T cells from lymph node of TCR transgenic TCRP1A B10D2 mice were sorted by flow cytometry (CD8+, CD3+, CD44-, Topro3-). For AdP1A samples, Naive CD8 T cells from lymph node of TCR transgenic TCRP1A B10D2 (Ly5.2) were transferred into Ly5.1 B10D2 mice. One day after adoptive transfer, mice were infected intra-dermally (ears) with the adenovirus AdP1At (coding for P1A, tha antigen recognized by TCRP1A CD8 T cells). Four days after infection draining lymph nodes were recovered and activated TCRP1A CD8 T cells were sorted by flow cytometry (CD8+, Ly5.2+, CD44+, Topro3-). For TILs samples, 2 to 3 melanoma tumor from TIRP mice (Huijbers Y, Cancer Res. 2006 Mar 15;66(6):3278-86) were pooled. After a ficoll, CD8 T cells were sorted by flow cytometry (CD3+, CD8+, Topro3-).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
SuperAmp RNA amplification was performed according to Miltenyi Biotec’s undisclosed procedure. Briefly, the amplification is based on a global PCR protocol using mRNA-derived cDNA. mRNA was isolated via magnetic bead technology. The integrity of the cDNA was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). The average length of the highly amplified cDNA products ranged between 200–1,000 bp. It’s noteworthy
|
| Label |
Cy3
|
| Label protocol |
250 ng of each of the cDNAs were used as template for Cy3 labeling which was performed according to Miltenyi Biotec’s undisclosed protocol.
|
| |
|
| Hybridization protocol |
The Cy3- labeled cDNAs were hybridized overnight (17 hours, 65°C) to an Agilent Whole Mouse Genome Oligo Microarrays 8 x 60K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min. The last washing step was performed with acetonitrile.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies).
|
| Description |
Naive TCR transgenic T cells (CD44 negative) sorted by flow cytometry
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers and calculates statistical confidences. For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolverâ gene expression data analysis system (Rosetta Biosoftware).
|
| |
|
| Submission date |
Dec 10, 2012 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Julien Maurizio |
| E-mail(s) |
maurizio@ciml.univ-mrs.fr
|
| Organization name |
INSERM
|
| Street address |
Centre d’Immunologie de Marseille-Luminy
|
| City |
Marseille |
| ZIP/Postal code |
13009 |
| Country |
France |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE42824 |
Molecular bases for tumor induced exhaustion in CD8 T cells |
|
