|
Status |
Public on Jun 01, 2013 |
Title |
Foxp3+ activated in vitro_Polysome-associated RNA_14 |
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: Polysome-associated 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 Polysome-associated RNA @52002900806729121711409035233638.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 |
|