|
| Status |
Public on Jun 16, 2015 |
| Title |
Day 16 post-LCMV infection (-Treg D8-16), biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
In vivo differentiated in the absence of regulatory T cells from Day 8-16
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: P14 cells from C57 Black 6 background
tissue: spleen
cell type: FACS purified GP33-specific CD8 T cells
treg depletion using dt tx: Tx on Days 8,10 and 12
days post-lcmv infection: Day 16
age: 8-10 weeks
|
| Treatment protocol |
First, CD8 T cells were enriched from the mouse splenocytes using a negative isolation protocol using a magnetic bead based kit from Stem cell technologies. The purified CD8 T cells (~70% pure) were fluorescently labeled for CD8 and Thy 1.1 for Fluorescence Activated Cell Sorting (FACS).Finally, CD8+ Db-GP33-specific T cells were FACS purified up to >98% purity for the next step of mRNA isolation. At least 10E6 purified CD8+ Db-GP33-specific T cells were used for each replicate from three distinct experiment to serve as the individual biological replicates.
|
| Growth protocol |
Chimeric FoXP3-DTR, mice adoptively transferred with 10E5 Db-GP33-specific naive P14 CD8 T cells, were infected with LCMV Armstrong. One set of the mice were treated with 20ng of diphtheria toxin (DT) per gram of mouse bodyweight on days 8, 10 and 12 post-infection to deplete regulatory T cells (Tregs). The control group of mice were treated with an equivalent amount of vehicle (PBS) on the indicated days. At day 16 post-infection, all mice were sacrificed and splenocytes were isolated for cell sorting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA from FACS purified CD8 T cells was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 2-4 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400. Hybridized cRNA was detected using streptavidin coupled to phycoerythrin
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| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A .Default values were used to grid images (.DAT) and generate .CEL and .CHP files and to generate gene expression values and ratios of gene expression between the hybridized samples
|
| Description |
Gene expression data from FACS purified GP33-specific CD8 T cells isolated at day 16 post-LCMV infection, Tregs depleted between day 8-16
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The .CEL files were analyzed using Genespring GX 11.5 software following RMA normalization.
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| |
|
| Submission date |
Apr 06, 2015 |
| Last update date |
Jun 16, 2015 |
| Contact name |
Surojit Sarkar |
| Organization name |
University of Washington School of Medicine
|
| Department |
Department of Pediatrics and Laboratory Medicine
|
| Lab |
Laboratory of T Cell Immunity to Pathogens and Cancer
|
| Street address |
1100 Olive Way, Suite 100
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98101 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE67593 |
Quiescence of Memory CD8+ T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4 |
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