|
| Status |
Public on Jun 07, 2010 |
| Title |
Macrophages day7 rep5 (MO MAC DC) |
| Sample type |
RNA |
| |
|
| Source name |
Monocyte-derived macrophages
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
sample type: primary cells
disease state: healthy donor
cell type: Monocyte-derived macrophages
|
| Treatment protocol |
Peripheral blood mononuclear cells were obtained from healthy donors by leukapheresis; elutriated monocytes were obtained form peripheral blood mononuclear cells by counter current elutriation; CD14+ sorted monocytes were isolated from peripheral blood mononuclear cells with the FACS_ARIA system
|
| Growth protocol |
Monocyte-derived dendritic cells (DC) were obtained by culturing elutriated monocytes with 20U/ml IL-4, 280U/ml GM-CSF and 10% FCS; monocyte-derived macrophages (MAC) were obtained by culturing elutriated monocytes with 2% AB serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA of the different cell types (monocytes, macrophages dendritic cells) was isolated after different time points (6h, 18h, 27h, 42h, 51h, 66h, day7) 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 for 16h at 65°C using a stringent protocol according to the manufacture's instructions (Agilent). 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; sample was hybridized to the Agilent GPL6848 (1x244K) expression array but adapted to the GPL6480 (4x44K) platform by GeneSpring version 7
|
| Data processing |
Data was processed using Feature Extraction Software (Agilent) and further analyzed using GeneSpring GX software version 10.0.2 (worksheet1: DC_kinetic) or version 7 (worksheet 2: MO_MAC_DC). Data were normalized to the 75 th percentile and baseline transformed to the median of freshly isolated monocytes. The sorted CD14+ cells (DC kinetic and MO MAC DC) and the elutriated monocytes (MO MAC DC) were considered as one monocyte population).
|
| |
|
| Submission date |
Dec 01, 2009 |
| Last update date |
Jun 07, 2010 |
| Contact name |
Maja Klug |
| E-mail(s) |
maja.klug@klinik.uni-r.de
|
| Organization name |
University Hospital Regensburg
|
| Department |
Hematology
|
| Street address |
Franz-Josef-Strauss-Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93042 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6848 |
| Series (1) |
| GSE19236 |
Expression analysis of dendritic cells, macrophages and monocytes |
|
