|
| Status |
Public on Apr 11, 2013 |
| Title |
MYD88-TRIF (dko) 5 (16h) - vs. MYD88-TRIF (dko) 5 (16h) LPS |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MyD88/TRIF (dko) 5 (16h) untreated
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: BMDM (bone marrow derived macrophages)
treatment: naive
genotype/variation: MyD88/Trif -/- double KO
time: 16h
|
| Treatment protocol |
Cells were stimulated with 200 ng/mL LPS (Salmonella minessota, ultrapure, Invivogen) in serum-free DMEM without phenolred or left untreated. At 1, 2, 4, 8 or 16 hours, cell supernatants were collected, passed through a 0.22 µm filter to remove detached cells, aliquotted and immediately frozen in liquid nitrogen.
|
| Growth protocol |
Bone marrow was collected from femur and tibia of age and gender matched mice of the indicated genotypes. Bone marrow cells were plated on sterile petridishes and incubated in DMEM containing 10% FCS, 5% equine serum, 10 mM HEPES, 1 mM pyruvate, 10 mM L-glutamine, and 20% M-CSF–conditioned medium at 37 °C and 7% CO2. M-CSF–conditioned medium was collected from an L929 M-CSF cell line. Bone marrow derived macrophages were harvested after 6 days and replated in tissue-culture treated dishes. Before the experiment, cells were washed once with serum-free DMEM without phenolred.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the TRIzol Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany) using Glycogen as carrier. Briefly, cells were resuspended in 1 ml TRIzol, shock frozen and stored at –80°C. Cells in Trizol were thawed and further processed for total RNA isolation as described by the manufacturer.
|
| Label |
Cy3
|
| Label protocol |
Total RNA labeling was performed with the two color Quick Amp Labeling Kit (Agilent Technologies).
|
| |
|
| Channel 2 |
| Source name |
MYD88/TRIF (dko) 5 (16h) LPS-treated
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cells: BMDM (bone marrow derived macrophages)
treatment: LPS
genotype/variation: MyD88/Trif -/- double KO
time: 16h
|
| Treatment protocol |
Cells were stimulated with 200 ng/mL LPS (Salmonella minessota, ultrapure, Invivogen) in serum-free DMEM without phenolred or left untreated. At 1, 2, 4, 8 or 16 hours, cell supernatants were collected, passed through a 0.22 µm filter to remove detached cells, aliquotted and immediately frozen in liquid nitrogen.
|
| Growth protocol |
Bone marrow was collected from femur and tibia of age and gender matched mice of the indicated genotypes. Bone marrow cells were plated on sterile petridishes and incubated in DMEM containing 10% FCS, 5% equine serum, 10 mM HEPES, 1 mM pyruvate, 10 mM L-glutamine, and 20% M-CSF–conditioned medium at 37 °C and 7% CO2. M-CSF–conditioned medium was collected from an L929 M-CSF cell line. Bone marrow derived macrophages were harvested after 6 days and replated in tissue-culture treated dishes. Before the experiment, cells were washed once with serum-free DMEM without phenolred.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the TRIzol Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany) using Glycogen as carrier. Briefly, cells were resuspended in 1 ml TRIzol, shock frozen and stored at –80°C. Cells in Trizol were thawed and further processed for total RNA isolation as described by the manufacturer.
|
| Label |
Cy5
|
| Label protocol |
Total RNA labeling was performed with the two color Quick Amp Labeling Kit (Agilent Technologies).
|
| |
|
| |
| Hybridization protocol |
After precipitation, purification and quantification, 1.25µg labeled samples were mixed and hybridized according to the supplier’s protocol (Agilent Technologies).
|
| Scan protocol |
Scanning of microarrays was performed with 5µm resolution and extended range using a G2565CA high resolution laser microarray scanner (Agilent Technologies).
|
| Description |
untreated vs LPS treated
|
| Data processing |
Microarray image data were extracted and analyzed with the Image Analysis / Feature Extraction software G2567AA (Version A.10.10.1, Agilent Technologies).
The normalized/processed data are included in the LogRatio column of the Agilent Feature Extraction files (ie, normalized log10 Cy5/Cy3 ratios).
Note: The NA probes of Platform GPL10333 are not present in the Agilent FE files.
|
| |
|
| Submission date |
Oct 11, 2012 |
| Last update date |
Apr 12, 2013 |
| Contact name |
Hans-Joachim Mollenkopf |
| E-mail(s) |
mollenkopf@mpiib-berlin.mpg.de
|
| Phone |
+49 30 28460 482
|
| Organization name |
Max-Planck-Institute for Infection Biology
|
| Lab |
Microarray/Genomics Core Facility
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10333 |
| Series (1) |
| GSE41490 |
Direct proteomic quantification of the secretome of activated immune cells |
|
