|
| Status |
Public on Aug 10, 2009 |
| Title |
Balbc IL4 4h rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Bone Marrow Macrophages
|
| Organism |
Mus musculus |
| Characteristics |
il4 stimulation: 4h
age: 8 to 12 weeks
sex: 2 males and 1 female
strain: Balb/cAnNCrl
tissue: Bone marrow
|
| Treatment protocol |
On day 7, cells were incubated for 18 h with or without IL4 (10 ng/ml). In the last 4 h of incubation, cells were incubated or not with LPS (10 ng/ml) for 4, 8 or 24 h (in the absence or presence of IL4).
|
| Growth protocol |
To generate bone marrow-derived macrophages (BMM), bone marrow cells were flushed from femurs and tibias of 8 to 12 weeks old mice using cold PBS. Cells from several animals were pooled and plated on bacteriological 100 mm square plastic plates (Bibby Sterilin, Staffordshire, UK Ltd) at 0.5 Mio cells/ml in 20 ml endotoxin-free RPMI 1640 medium (Biochrom KG, Berlin, Germany) supplemented with glutamine, vitamins, pyruvate, nonessential amino acids, β-mercaptoethanol (all purchased from Invitrogen, Germany), 10% FCS (PAA Laboratories GmbH, Austria), and 200 ng/ml human rCSF1 (Cetus) per plate for 5 days. Complete medium was replaced on day 5, cells were harvested on day 6 and seeded with rCSF1 at a density of 10 Mio cells/10 ml medium on 10 cm tissue culture dishes (Falcon).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected, pelleted and used for total RNA preparation with the RNeasy midi kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Agilent’s Low RNA Input Linear Amplification One-Color kit (Agilent Technologies, Inc. Wimmington, DE, USA) was used to label the target RNA for hybridization following the manufacturer instructions. Briefly, 1 µg total RNA was reverse transcribed using a T7 promoter Primer. The resulting cDNAs were then in vitro transcribed followed by reverse transcription in the presence of Cyanin 3-CTP.
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| |
|
| Hybridization protocol |
Labelled cRNA was then purified using Qiagen’s RNease mini spin columns (Qiagen) and 1.65 µg from each sample were fragmented for 30 min and mixed with a 2xGExHybridization Buffer HI-RPM according to the manufacturer’s instructions (Agilent Technologies). The resulting mixture was applied to a Whole Mouse Genome (4x44k) Oligo Microarray (Agilent Technologies Inc.) and the hybridization followed at 65°C for 17 h.
|
| Scan protocol |
Arrays were then washed and scanned according to Agilent protocols with the Agilent Scanner.
|
| Description |
Gene expression after 4h IL-4 stimulation
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb 97 and Grid: 014868_D_20060807) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Extracted data were further processed with GeneSpring GX 7.3.1. Data Values below 10.0 were set to 10.0. Each measurement was divided by the 50th percentile of all measurements in that sample. The percentile was calculated using only genes marked present. Each gene was divided by the median of its measurements in all samples. If the median of the raw values was below 0 then each measurement for that gene was divided by 0 if the numerator was above 0, otherwise the measurement was thrown out. Median polishing was done, where each chip was normalized to its median and each gene was normalized to its median. These normalizations were repeated until the medians converged. Prior to median polishing, raw values below 10 were replaced with 10. All genes except those marked absent were used in this calculation. Specific samples were normalized to one another: sample(s) 1-12 were normalized against the median of the control sample(s) 7, 10. Each measurement for each gene in those specific samples was divided by the median of that gene's measurements in the corresponding control samples.
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| |
|
| Submission date |
Jan 29, 2009 |
| Last update date |
Aug 10, 2009 |
| Contact name |
Carol El Chartouni |
| Organization name |
University Hospital Regensburg
|
| Department |
Hematology and oncology
|
| Lab |
PD.Dr. Rehli
|
| Street address |
Franz-Josef-Strauß Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE14644 |
IL-4-primed bone marrow macrophages isolated from C57Bl6/J or Balb/cAnNCrl mice |
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