|
| Status |
Public on May 22, 2011 |
| Title |
macrophage_wildtype_E.coli_3h_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
bone marrow derived macrophages from WT (C57BL6/J) mice, stimulated with E.coli for 3hrs
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
gender: Female
age: 5-6 weeks
genotype/variation: wild type
cell type: bone marrow macrophage (BMM)
protocol: E.coli stimulation
time: 3h
|
| Treatment protocol |
The cultured bone marrow derived macrophages (BMMs) were detached and re-plated 4 hours prior to the experiment. At the start of the experiment, BMMs were incubated with viable E. coli for the 1, 3 or 6 hours. Bacteria were used at a multiplicity of infection (MOI) of 20. All experiments were carried out in antibiotic free 'complete medium'. One hour after addition of viable bacteria, penicillin (100μg/ml) and streptomycin (100μg/ml) were added to the media in order to kill any remaining extracellular bacteria. For transcriptional analysis three independent experiments were performed. We compared this approach to washing the cells and replacing the antibiotic free medium with penicillin/streptomycin containing medium after one hour and found no differences with regards to the cellular responses measured.
|
| Growth protocol |
Murine macrophages, derived from the bone marrow of either C57BL/6J, or Trif-/- mice, were cultured in RPMI 1640 supplemented with macrophage colony-stimulating factor (M-CSF) and 10% FBS, plus 100μg/ml penicillin, 100μg/ml streptomycin, 10mM HEPES and 1nM sodium pyruvate (all obtained from Sigma-Aldrich).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the BMMs using the RNeasy kit (QIAGEN). RNA from RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands) with 6000 Nano Chips. RNA was judged as suitable only if samples showed intact bands of 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RNA integrity number (RIN) above 8.0.
|
| Label |
biotin
|
| Label protocol |
The Affymetrix GeneChip WT Sense Target Labeling and Control Reagents kit (P/N 900652) was used for the preparation of labelled cDNA from 100ng of total RNA without rRNA reduction. A detailed description can be found in the User Manual, Chapter 3 (P/N 701880, revision 5).
|
| |
|
| Hybridization protocol |
The Affymetrix GeneChip Mouse Gene 1.1 ST 24-array plate consists of 24 single Gene 1.1 ST arrays arranged into the standard 96 well plate format. Array hybridization, washing and scanning were performed on a GeneTitan Instrument according to the manufacturer’s recommendations.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneTitan instrument.
|
| Description |
WT_Ecoli3h_3
G004_C09_19_117-3_WT_Ecoli3h.CEL
|
| Data processing |
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.14.0).
|
| |
|
| Submission date |
Mar 15, 2011 |
| Last update date |
May 22, 2011 |
| Contact name |
Guido Hooiveld |
| E-mail(s) |
guido.hooiveld@wur.nl
|
| Organization name |
Wageningen University
|
| Department |
Div. Human Nutrition & Health
|
| Lab |
Nutrition, Metabolism & Genomics Group
|
| Street address |
HELIX, Stippeneng 4
|
| City |
Wageningen |
| ZIP/Postal code |
NL-6708WE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11533 |
| Series (1) |
| GSE27960 |
Sensing prokaryotic mRNA signifies microbial viability and promotes immunity |
|
