|
| Status |
Public on Jun 01, 2013 |
| Title |
Foxp3- activated in vitro_Cytoplasmic RNA_11 |
| Sample type |
RNA |
| |
|
| Source name |
Cells isolated from lymph nodes and spleens
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Foxp3-
condition: Activated in vitro for 36 hrs with plate bound CD3 and CD28 antibodies (BD Bioscience) (5µg/ml) in the presence of recombinant hIL-2 ([100U/ml]
isolated rna: Cytoplasmic RNA
|
| Treatment protocol |
For the naïve cells all buffers and media were supplemented with cycloheximide (Sigma, St. Louis, MO) (100µg/ml). For the activated samples cells were activated for 36h with plate bound CD3 and CD28 antibodies (BD Bioscience) (5µg/ml) in the presence of recombinant hIL-2 ([100U/ml]
|
| Growth protocol |
Cells isolated from lymph nodes and spleens were stained with PE conjugated CD4 antibody (GK1.5, eBioscience, San Diego, CA) and MACS purified. Thereafter TFoxp3+ and TFoxp3- cells were sorted based on CD4 and GFP-Foxp3 expression using a FACSAria to obtain cell populations of high purity (>97%). For the activated samples cells were activated for 36h with plate bound CD3 and CD28 antibodies (BD Bioscience) (5µg/ml) in the presence of recombinant hIL-2 ([100U/ml]
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cytosolic and polysome-associated RNA were prepared directly ex vivo or post-activation in vitro as described previously (Larsson O, 2007 ancer Res 67: 6814-6824). For cells isolated directly ex vivo, RNA from two experiments was pooled for each sample.
|
| Label |
biotin
|
| Label protocol |
Ovation Pico WTA system (NuGEN) according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Samples were hybridized with the Affymetrix GeneChip Mouse Genome 430 2.0 Array according the instructions of the manufacturer
|
| Scan protocol |
Arrays were scanned using the GeneArray Scanner 3000 according to the instructions from the manufacturer.
|
| Description |
Gene expression data from Foxp3- cells Activated in vitro for 36 h using RNA isolated from Cytoplasmic RNA
@52002900806613121411408986822851.CEL
|
| Data processing |
Data were extracted and normalized using a custom CDF and Robust Multi-array Average (RMA) in R (r-project.org) with default settings.
|
| |
|
| Submission date |
Mar 21, 2013 |
| Last update date |
Jun 01, 2013 |
| Contact name |
Ola Larsson |
| E-mail(s) |
ola.larsson@ki.se
|
| Phone |
+46 (0)8 517 73280
|
| Organization name |
Karolinska Institutet
|
| Department |
Department of oncology-pathology
|
| Street address |
CCK, R8:01
|
| City |
Stockholm |
| ZIP/Postal code |
171 76 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL7546 |
| Series (1) |
| GSE45401 |
Distinct translational control in CD4+ T-cell subsets |
|
