|
| Status |
Public on Jan 01, 2011 |
| Title |
Macrophage Ctrl 12h Expression (Cy3) |
| Sample type |
RNA |
| |
|
| Source name |
thioglycollate-elicited peritoneal macrophages
|
| Organism |
Mus musculus |
| Characteristics |
cell type: thioglycollate-elicited peritoneal macrophages
treatment: dmso for 12 hours
strain: C57BL/6
|
| Growth protocol |
CD43-negative splenic B cells were isolated by magnetic depletion of CD43- and CD11b-expressing cells using MACS (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Peritoneal macrophages were harvested by peritoneal lavage with 10 ml ice-cold PBS 3 days after peritoneal injection of 3 ml thioglycollate broth. Peritoneal cells were washed once with PBS, and seeded in 10% fetal calf serum (FCS)/DMEM containing 100 U/ml penicillin/streptomycin in tissue culture-treated petri dishes overnight. Non-adherent cells were washed off with room temperature PBS. Bone marrow was harvested and washed once with PBS, seeded/cultured in DMEM containing 20 % FCS, 30 % L-cell conditioned media (as source of macrophage colony stimulating factor, M-CSF), and 100 U/ml penicillin/streptomycin in non-tissue culture-treated petri dishes. After 6-8 days of culture, non-adherent cells were washed off with room temperature PBS and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in RPMI 1640 containing 10% FCS, 100 U/ml penicillin/streptomycin, and 10ng/ml M-CSF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 5 Mio cells on Rneasy columns (Qiagen) with on-column DNase digestion following the manufacturer's instructions. RNA quality was assessed on an Agilent 2100 BioAnalyzer.
|
| Label |
Cy3
|
| Label protocol |
Total RNA (250 ng) of two biological replicates was amplified and labeled using the Agilent Quick-Amp labeling kit according to the manufacturer's instructions.
|
| |
|
| Hybridization protocol |
4x 44 k Agilent arrays were hybridized with 825 ng of each cDNA using the manufacturer's hybridization kit according to the instructions.
|
| Scan protocol |
Scanned on an Agilent Technologies Scanner using the extended dynamic range scan mode with a 5 µm pitch.
Images were quantified using Agilent Feature Extraction Software (version 9.5.3.1).
|
| Description |
Macrophage Expression in response to mock treatment at 12 hours
Raw data file: US22502657_251486819799_S01_GE2-v5_95_Feb07_1_3.txt
|
| Data processing |
Agilent Feature Extraction Software (v 9.5.3.1) was used for background subtraction and LOWESS normalization. Data was further normalized between arrays using a multi-loess technique described in Corbeil et al (Journal of Molecular Endocrinology (2004) 33:1–9).
Dual-channel hybridization was analyzed as single-channel (ratios were not used). Each channel of biological replicates is therefore represented as a single Sample and the same raw data file is linked to two Sample records.
|
| |
|
| Submission date |
Aug 13, 2010 |
| Last update date |
Jan 01, 2011 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
|
| Organization name |
University of California, San Diego (UCSD)
|
| Department |
Medicine
|
| Street address |
9500 Gilman Dr. MC 0640
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE23620 |
Mechanisms Establishing TLR4-Responsive Activation States of Inflammatory Response Genes (Agilent expression data) |
| GSE23622 |
Mechanisms Establishing TLR4-Responsive Activation States of Inflammatory Response Genes |
|
