|
| Status |
Public on Dec 31, 2012 |
| Title |
unstimulated vs stimulated 96_FBR_Low 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
DN T-cells
|
| Organism |
Cercocebus atys |
| Characteristics |
cd4 status: Low
treatment: Unstimulated
cell type: Double Negative (CD4-/CD8-) T-cells
infection: SIV
|
| Treatment protocol |
1 million purified T cells were stimulated with anti-CD3 (3mg/ml) and anti-CD28 (1mg/ml) in 200ul of complete RPMI at 37oC for 4 hrs.
|
| Growth protocol |
Pure population of double negative and CD4 T cells were isolated by a step-wise magnetic bead isolation (Miltenyi) using bead specific for non-human primate antigens. Briefly, we stained PBMC with CD4-Microbead to isolate CD4 cells and treated the flow-through with CD8-PE and CD16-PE followed by anti-PE Microbeads for CD8 and CD16 depletion. This CD4-CD8-CD16- flow-through was then positive selected for CD3+ cells with CD3-Biotin and anti-biotin Microbead to obtain, CD3+CD4-CD8- (double negative T cells). Purity was checked after each isolation and further experiments were carried out only with >98% pure populations. Cells were rested overnight prior to functional analyses.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected post-stimulation and immediately lysed in RLT buffer (Qiagen) for RNA extraction. RNA was isolated using RNeasy mini kit (Qiagen) and genomic DNA was removed on-column.
|
| Label |
Cy3
|
| Label protocol |
cRNA was synthesized from total RNA and Cy3/Cy5 labeled using the Agilent Low Input Quick Amp Labeling procedure, as per the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
DN T-cells
|
| Organism |
Cercocebus atys |
| Characteristics |
cd4 status: Low
treatment: Stimulated with CD3/CD28 antibodies
cell type: Double Negative (CD4-/CD8-) T-cells
infection: SIV
|
| Treatment protocol |
1 million purified T cells were stimulated with anti-CD3 (3mg/ml) and anti-CD28 (1mg/ml) in 200ul of complete RPMI at 37oC for 4 hrs.
|
| Growth protocol |
Pure population of double negative and CD4 T cells were isolated by a step-wise magnetic bead isolation (Miltenyi) using bead specific for non-human primate antigens. Briefly, we stained PBMC with CD4-Microbead to isolate CD4 cells and treated the flow-through with CD8-PE and CD16-PE followed by anti-PE Microbeads for CD8 and CD16 depletion. This CD4-CD8-CD16- flow-through was then positive selected for CD3+ cells with CD3-Biotin and anti-biotin Microbead to obtain, CD3+CD4-CD8- (double negative T cells). Purity was checked after each isolation and further experiments were carried out only with >98% pure populations. Cells were rested overnight prior to functional analyses.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected post-stimulation and immediately lysed in RLT buffer (Qiagen) for RNA extraction. RNA was isolated using RNeasy mini kit (Qiagen) and genomic DNA was removed on-column.
|
| Label |
Cy5
|
| Label protocol |
cRNA was synthesized from total RNA and Cy3/Cy5 labeled using the Agilent Low Input Quick Amp Labeling procedure, as per the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Labeled cRNA was hybridized to the Rhesus Macaque (V2) Gene Expression 4x44K Microarray (Agilent) in the appropriate combinations, including dye-flip and self versus self controls.
|
| Scan protocol |
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Description |
96_FBR_Low
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
After scanning the arrays, data was background subtracted and the probe signal normalized both within the array and between the arrays, using the limma package in Bioconductor.
|
| |
|
| Submission date |
Sep 11, 2012 |
| Last update date |
Dec 31, 2012 |
| Contact name |
Ramsey Saleem |
| E-mail(s) |
ramsey.saleem@seattlebiomed.org
|
| Phone |
2062567446
|
| Organization name |
Seattle Biomedical Research
|
| Street address |
307 Westlake Ave N
|
| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98199 |
| Country |
USA |
| |
|
| Platform ID |
GPL16026 |
| Series (1) |
| GSE40781 |
CD3/CD28 Stimulated versus unstimulated Double-negative T Cells From Sooty Mangabeys |
|
