|
| Status |
Public on Oct 08, 2009 |
| Title |
DC control 18h rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Bone-marrow derived dendritic cells, control, 18h
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
treatment: Control
time: 18 hours
|
| Treatment protocol |
Immature DC (2 x 10^6 cells/ml) were resuspended in fresh medium supplemented with 10 ng/ml GM-CSF, and 500 µl/well were seeded in 48-well tissue culture plates (Nunc, Roskilde, Denmark). L. acidophilus NCFM were suspended in medium and added (100 µl/well) in a final concentration of 10 μg/ml and the cell cultures were incubated at 37ºC in 5 % CO2 for the duration of the treatment.
|
| Growth protocol |
Bone marrow from wild type (WT) C57BL/6 mice was flushed out from the femur and tibia and washed twice in sterile PBS. 3 x 105 bone marrow cells were seeded into Petri dishes in 10 ml RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 10% (v/v) heat-inactivated fetal calve serum supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (4 mM), 50 µm 2-mercaptoethanol (all purchased from Cambrex Bio Whittaker) and 15 ng/ml murine GM-CSF (harvested from a GM-CSF transfected Ag8.653 myeloma cell line). The cells were incubated for 8 days at 37 ºC in 5% CO2 humidified atmosphere. On day 3, 10 ml of complete medium containing 15 ng/ml GM-CSF was added. On day 6, 10 ml were removed and replaced by fresh medium. Non-adherent immature DC were harvested on day 8.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Murine DC, harvested at various stimulation time points, were homogenised by QIAshredder (Qiagen, Ballerup, Denmark), and RNA was extracted using the RNeasy Plus Mini Kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
1 μg RNA per stimulation condition was converted into cDNA, and biotin-labeled aRNA was synthesized using the MessageAmpTM II-Biotin Enhanced Kit (Ambion, Austin, TX, USA) according to the manufacturers instructions.
|
| |
|
| Hybridization protocol |
The arrays were stained and washed according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA) using the GeneChip Fluidics Station 450
|
| Scan protocol |
The arrays were scanned according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA) using Affymetrix GeneChip Scanner 7G.
|
| Description |
none
|
| Data processing |
The microarray data was analyzed using R and Bioconductor (Gentleman et al. 2004). Raw probe intensities were normalized using qspline and expression index calculations were performed using rma (Irizarry et al. 2003;Workman et al. 2002). For statistical testing ANOVA was performed using stimulation time as factor where all untreated samples were treated as one group.
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| |
|
| Submission date |
Oct 07, 2009 |
| Last update date |
Oct 07, 2009 |
| Contact name |
Simon Rasmussen |
| E-mail(s) |
[email protected]
|
| Phone |
+45 4525 6148
|
| Organization name |
Technical University of Denmark
|
| Department |
Department of Systems Biology
|
| Lab |
Center for Biological Sequence Analysis
|
| Street address |
Kemitorvet, Building 208
|
| City |
Kongens Lyngby |
| ZIP/Postal code |
DK-2800 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE18460 |
Lactobacillus acidophilus induces virus immune defense genes in murine dendritic cells by a TLR-2 dependent mechanism |
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