|
| Status |
Public on Dec 31, 2019 |
| Title |
Donor 1: M2c MФ |
| Sample type |
RNA |
| |
|
| Source name |
Human CD14(+) monocyte-derived macrophages
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
individual: Donor 1
origin: CD14(+) monocyte-derived macrophages
cell type: M2c MФ
|
| Treatment protocol |
As described in Sample Characteristics
|
| Growth protocol |
As described in Sample Characteristics
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA of the different cell types was isolated using the RNeasy Plus 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, 500 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 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 |
Isolated monocytes were cultured for 6 days at a density of 1x10^5 cells/cm^2 in Cell+ coated plates with RPMI medium containing 20% FCS, 100 ng/ml rhM-CSF, P/S and 2mM GlutaMax. The cells were then stimulated with dexamethasone at 10(-7) M for a further 24 hours in medium containing 5% FCS in medium containing 5% FCS.
|
| Data processing |
Data was processed using Feature Extraction Software (Agilent) and further analyzed using GeneSpring GX software version 10. Raw data was normalized by a 75 percentile shift, log2-transformed and shifted to the median of all samples.
|
| |
|
| Submission date |
Jul 22, 2009 |
| Last update date |
Dec 31, 2019 |
| Contact name |
James Alexander Hutchinson |
| E-mail(s) |
james.hutchinson@klinik.uni-regensburg.de
|
| Organization name |
University Hospital Regensburg
|
| Department |
General Surgery
|
| Lab |
Transplant Immunology
|
| Street address |
Franz-Josef-Strauss-Allee-11
|
| City |
Regensburg |
| State/province |
Bavaria |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
|
