|
| Status |
Public on May 11, 2009 |
| Title |
Treg_culture_CD4+CD25+CD45RA+_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Treg expansion culture
|
| Organism |
Homo sapiens |
| Characteristics |
Treg culture CD4+CD25+CD45RA+
|
| Treatment protocol |
Treg and Tconv cell FACS sorting and expansion culture was performed as previously described by Hoffmann et al. 2004 (Blood. Aug 1;104(3):895-903). Cell-culture was performed according to Hoffmann et al. 2006 (Blood. Dec 15;108(13):4260-7).
|
| Growth protocol |
Treg and Tconv cell isolation and expansion culture was performed as previously described by Hoffmann et al. 2004 (Blood. Aug 1;104(3):895-903). Cell-culture was performed according to Hoffmann et al. 2006 (Blood. Dec 15;108(13):4260-7).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA of the different cell types was isolated using the RNeasy Kit (Qiagen). RNA concentration was measured with a ND-1000 Spectrophotometer (NanoDrop, Thermo Fisher Scientific) and quality was controlled on agarose gels or using the Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Labeling and hybridization were performed using the Agilent Gene Expression system according to the manufacturer’s instructions. In brief, 200 ng to 1000 ng of high-quality RNA were amplified and Cyanine 3-CTP labeled with the One Color Low RNA Input Linear Amplification Kit (Agilent). Labeling efficiency was controlled using the NanoDrop spectrophotometer.
|
| |
|
| Hybridization protocol |
Samples were hybridized using a stringent protocol according to the manufacture's instruction. 1.65 µg labeled cRNA was supplemented with 11 µl Agilent 10x blocking agent, 2.2 µl Agilent 25x fragmentation buffer and nuclease-free water up to 55 µl final volumen. The sample was incubated at 60°C for 30 minutes, supplemented with 55 µl 2x hybridisation buffer and 100 µl of the mix was hybridisized for 16h at 65°C using an SureHyb chamber and an Agilent hybridization oven. Arrays were disassembled and washed for 1 minute in Agilent gene Expression (GE) wash buffer 1 followed by washing 1 minute in prewarmed (37°C) GE wash buffer 2.
|
| Scan protocol |
Images were scanned immediately after washing using a DNA microarray scanner (Agilent) at 5 µm resolution.
|
| Description |
gender: male
|
| Data processing |
Data was processed using Feature Extraction Software (Agilent) and further analyzed using GeneSpring GX software version 7.
|
| |
|
| Submission date |
Dec 30, 2008 |
| Last update date |
May 11, 2009 |
| Contact name |
Christian Schmidl |
| E-mail(s) |
[email protected]
|
| Organization name |
Rengensburg Center for Interventional Immunology
|
| Lab |
Schmidl Lab
|
| Street address |
Franz-Josef-Strauss-Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE14232 |
Transcriptome analysis of freshly sorted and expanded regulatory and conventional T cells |
| GSE14281 |
Regulatory and conventional T-cells |
|
