|
| Status |
Public on Sep 20, 2007 |
| Title |
Gr1-_2h LM infected_2_peritoneal lavage |
| Sample type |
RNA |
| |
|
| Source name |
infected peritoneal cells, timepoint 2h
|
| Organism |
Mus musculus |
| Characteristics |
Six-weeks old (C57Bl6, Cx3cr1gfp/+) mice
infected with 1x104 of L. monocytogenes (EGDe strain) in exponential growth phase
timepoint:2h
Gr1- monocytes purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1-, gfphigh
|
| Treatment protocol |
Cells were directly sorted in the SuperAmp Lysis Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) using a FACS Aria cell-sorter (BD biosciences). Cell samples (1.103 cells per experimental point) were lysed using SuperAmp Lysis Buffer following manufacturer’s instructions
|
| Growth protocol |
Mice were intraperitonealy infected with 104 L. monocytogenes (EGDe strain) in exponential growth phase (bacteria were grown in BHI at 108/ml, and diluted 10.000x in PBS immediately before injection).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
SuperAmplification was performed according to Miltenyi Biotec’s undisclosed protocol. Briefly, the amplification is based on a global PCR protocol of mRNA-derived cDNA. mRNA was isolated via magnetic bead technology.
|
| Label |
Cy3
|
| Label protocol |
250 ng of each of the cDNAs were used as template for Cy3 labeling which was performed according to Miltenyi Biotec’s undisclosed protocol.
|
| |
|
| Hybridization protocol |
Cy3 labeled cDNA in hybridization buffer was hybridized overnight (17 hours, 65°C) to an Agilent Whole Mouse Genome Oligo Microarrays (44K) using Agilent’s recommended hybridization chamber and oven.Finally, microarrays were washed once with 6x SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min followed by a second wash with 0.06x SSPE containing 0.005% N-lauroylsarcosine for 1 min and a final wash step with acetonitrile for 30 sec. All washing steps were performed at room temperature.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies, Palo Alto, USA).
|
| Description |
Cells were directly sorted in the SuperAmp Lysis Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) using a FACS Aria cell-sorter (BD biosciences).
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files
|
| |
|
| Submission date |
Jun 26, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Silvia Rueberg |
| E-mail(s) |
silvia@miltenyibiotec.de
|
| Organization name |
Miltenyi Biotec GmbH
|
| Department |
Genomic Services
|
| Street address |
Friedrich-Ebert-Str. 68
|
| City |
Bergisch Gladbach |
| ZIP/Postal code |
51429 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE8294 |
Microarray analysis of monocytes recruited in the peritoneum during experimental infection with Listeria monocytogenes |
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