|
| Status |
Public on Jan 01, 2009 |
| Title |
Averaged 2021_1 ... 2021_4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PBM
|
| Organism |
Mus musculus |
| Characteristics |
Cells: Peripheral Blood Monocytes
Isolation: FACS from broncho-alvoelar lavage (24h post challange)
Animals: CX3CR1GFP/GFP mice on a mixed C57BL/6 x 129/Ola background
Age: 8 - 12 weeks
Gender: Mixed
Treatment: i.t. application of 50 µg TLR2 ligand Pam3CSK4
|
| Biomaterial provider |
Maciej Cabanski
|
| Treatment protocol |
Mice were anesthetized with tetrazoline hydrochloride and ketamine, and the trachea was exposed. Subsequently, catheter (Abbot, Wiesbaden, Germany) was inserted into trachea and Pam3CSK4 (50 µg/mouse dissolved in total volume of 60 µl) was installed under stereomicroscopic control (Leica MS5, Wetzlar, Germany). After installation, wounds were closed and mice were allowed to recover with free access to food and water.
24 h post-challenge, mice were sacrificed with an overdose of isoflurane (Forene; Abbott, Wiesbaden, Germany) and blood and BAL fluid (BALF) collection was done as describe previously (Srivastava 2005 J Immunol 175(3):1884-93; Steinmuller 2006 Respir Res 7:30).
Blood was collected from the vena cava inferior. The lysis of red blood cells was performed using ammonium chloride solution.
Prior to cell sorting, samples were filtered through a 40 µm cell strainer.
|
| Growth protocol |
Mice were kept under specified pathogen free conditions with free access to water and food.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was isolated from 30.000-40.000 freshly sorted GFP-positive MPs using the RNeasy Micro kit. RNA was eluted in 12 μl RNase free H2O. RNA was quantified spectrophotometrically (Nanodrop ND-100) and the quality was assessed by the 18S/28S-rRNA bands in capillary electrophoresis (Bioanalyzer 2100, Agilent Biosystems).
|
| Label |
Cy3,Cy5
|
| Label protocol |
Purified total RNA was amplified and Cy-labeled using the SMART™ kit (Clontech BD Healthcare) following the kit instructions with 24 amplification cycles. Monoreactive CyDyes (Amersham) were coupled in subsequent 4 cycles.
|
| |
|
| Channel 2 |
| Source name |
AM
|
| Organism |
Mus musculus |
| Characteristics |
Cells: Alveolar Macrophages
Isolation: FACS from broncho-alvoelar lavage (BAL) (24h post challenge)
Animals: CX3CR1GFP/GFP mice on a mixed C57BL/6 x 129/Ola background
Age: 8 - 12 weeks
Gender: Mixed
Treatment: i.t. application of 50 µg TLR2 ligand Pam3CSK4
|
| Treatment protocol |
Mice were anesthetized with tetrazoline hydrochloride and ketamine, and the trachea was exposed. Subsequently, catheter (Abbot, Wiesbaden, Germany) was inserted into trachea and Pam3CSK4 (50 µg/mouse dissolved in total volume of 60 µl) was installed under stereomicroscopic control (Leica MS5, Wetzlar, Germany). After installation, wounds were closed and mice were allowed to recover with free access to food and water.
24 h post-challenge, mice were sacrificed with an overdose of isoflurane (Forene; Abbott, Wiesbaden, Germany) and blood and BAL fluid (BALF) collection was done as describe previously (Srivastava 2005 J Immunol 175(3):1884-93; Steinmuller 2006 Respir Res 7:30).
Blood was collected from the vena cava inferior. The lysis of red blood cells was performed using ammonium chloride solution.
Trachea was exposed and a small incision was made to insert a shortened 21-gauge cannula connected to a 1-ml insulin syringe, followed by repeated i.t. instillations of 0.5 ml aliquots of PBS (pH 7,2, supplemented with 2 mM EDTA). BAL was performed until a total volume of 5 ml was recovered. BALF was spun for 10 min at 1400 rpm at 4°C and cells were dissolved in 1 ml of RPMI 1640 medium containing 10 % FCS and L-glutamine. Recovered BAL and blood cells were pooled from 10-12 CX3CR1GFP/+ mice to obtain sufficient cell numbers for flow sorting.
Prior to cell sorting, samples were filtered through a 40 µm cell strainer.
|
| Growth protocol |
Mice were kept under specified pathogen free conditions with free access to water and food.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was isolated from 30.000-40.000 freshly sorted GFP-positive MPs using the RNeasy Micro kit. RNA was eluted in 12 μl RNase free H2O. RNA was quantified spectrophotometrically (Nanodrop ND-100) and the quality was assessed by the 18S/28S-rRNA bands in capillary electrophoresis (Bioanalyzer 2100, Agilent Biosystems).
|
| Label |
Cy3,Cy5
|
| Label protocol |
Purified total RNA was amplified and Cy-labeled using the SMART™ kit (Clontech BD Healthcare) following the kit instructions with 24 amplification cycles. Monoreactive CyDyes (Amersham) were coupled in subsequent 4 cycles.
|
| |
|
| |
| Hybridization protocol |
Labeled cDNAs were hybridized in Agilent in-situ hybridization buffer overnight at 60°C. Slides were mounted in Agilent hybridization chambers that were kept rotating at 10 rpm in an hybridization oven. Hybridization and subsequent washing and drying of the slides were performed following the Agilent hybridization protocol.
|
| Scan protocol |
Dried slides were scanned using the GenePix 4100A scanner (Axon Instruments). Photomultiplier tube gains were adjusted to get about 1% spots with saturated pixels and similar intensity histograms in both channels.
|
| Description |
no additional information required
|
| Data processing |
Data were evaluated using the R software (http://www.R-project.org) and the limma package from BioConductor (Gentleman R 2004 Genome Biol 5(10):R80, Smyth GK 2004 Stat Appl Genet Mol Biol 3:Article3). The spots were weighted for subsequent analyses according to the spot intensity, homogeneity, and saturation. The spot intensities were corrected for the local background using the method of Edwards with an offset of 64 to stabilize the variance of low-intensity spots. The M/A data were LOESS normalized before averaging. Genes were ranked for differential expression using a moderated t-statistic. Candidate lists were created by adjusting the false-discovery rate to 10 %.
|
| |
|
| Submission date |
Jul 03, 2008 |
| Last update date |
Aug 05, 2008 |
| Contact name |
Jochen Wilhelm |
| Organization name |
Uniklinikum Giessen
|
| Department |
ECCPS Microarray Unit
|
| Street address |
Gaffkystrasse 10
|
| City |
Giessen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE11978 |
Gene expression profiling of peripheral blood and alveolar recruited mononuclear phagocytes in Pam3CSK4 treated mice |
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