|
| Status |
Public on Mar 05, 2009 |
| Title |
BMDCs_PBS_ 6h_2 |
| Sample type |
RNA |
| |
|
| Source name |
bone marrow derived dendritic cells
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
age: 6 - 8 weeks
|
| Treatment protocol |
Immature DCs (1 × 10^6 cells/ml) were incubated with either 300 µg/ml γ-PGA NPs, 1 µg/ml LPS, or 300 µg/ml unparticulate γ-PGA. The cultures were incubated for 6 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Growth protocol |
Femurs and tibias were removed from mice and purified from the surrounding muscle tissues. Both ends of the bones were cut with scissors, and the marrow was flushed with PBS using a syringe with a 26-gauge needle. The suspension was passed through a 50-mm cell strainer. Bone marrow cells (2.5 × 10^6 cells/10 ml) were suspended in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal bovine serum, 100 U/ml penicillin G, 100 µg/ml streptomycin, 2-mercaptoethanol, and 20 ng/ml recombinant GM-CSF (PeproTech) and placed into a Petri dish (InaOptica). Fresh culture medium was added to the dish on day 3. On day 7, non-adherent or loosely adherent cells were harvested and used as immature DCs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the cells with RNeasy (Qiagen). The quality of the total RNA was examined by 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
RNA (500 ng) was converted to cDNA with Molony murine leukemia virus RT and T7 promoter primer. The cDNA was transcribed and amplified with T7 RNA polymerase to produce the cRNA labeled with cyanine 3. The cyanine 3-labeled cRNA was purified with RNeasy and examined for its concentration and labeling quality by a spectrophotometer.
|
| |
|
| Hybridization protocol |
The cRNA was fragmented and hybridized to Agilent whole mouse genome oligonucleotide microarray (4 × 44K slide format) at 37 ºC for 17 hours.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides
|
| Description |
Gene expression after 6hr in PBS-treated mouse bone marrow derived dendritic cells
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1.3.1 (Agilent) using default parameters (protocol GE1-v5_91_0806 and Grid: 014868_D_20060807) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Mar 03, 2009 |
| Last update date |
Mar 04, 2009 |
| Contact name |
Takayuki Hamasaki |
| E-mail(s) |
t-hamask@m2.kufm.kagoshima-u.ac.jp
|
| Phone |
+81-99-275-5931
|
| Organization name |
Kagoshima University
|
| Department |
Graduate School of Medical and Dental Sciences
|
| Lab |
Center for Chronic Viral Diseass
|
| Street address |
8-35-1 Sakuragaoka
|
| City |
Kagoshima |
| State/province |
Kagoshima |
| ZIP/Postal code |
890-9544 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE15087 |
Comparison of gene expression in BMDCs stimulated with gamma-PGA nanoparticles, LPS and unparticulate gamma-PGA |
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