|
| Status |
Public on Dec 08, 2015 |
| Title |
WT2M2, biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Bone Marrow Derived Macrophages
|
| Organism |
Mus musculus |
| Characteristics |
cell type: BMDM
treatment: alternatively activated (M2 condition) with IL-4
|
| Treatment protocol |
On day 7 in culture the cells were washed, counted and replated in DMEM media (without L929 supernatant) at a density of 6-8^6 cells/well (6-well plate, Falcon polystyrene). Cells were classically activated (M1 condition) with LPS (100 ng/ml, Sigma-Aldrich) + IFN-γ (20ng/mL, E-bioscience, San Diego, CA) or alternatively activated (M2 condition) with IL-4 (20ng/mL, E-bioscience) or received media alone (M0 condition). Cells were harvested at 24 hours post-stimulation, by washing in phosphate buffered saline (PBS) before cell lysis in miRVana Lysis buffer (Life Technologies) for total RNA isolation.
|
| Growth protocol |
To generate BMDM, the bone marrow cells from femurs and tibias from mice were harvested and cultured. Briefly, isolated cells were incubated in Dulbecco’s Modified Eagle Media (DMEM, Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Grand Island, NY)), 1% penicillin/streptomycin, 1% glutamine, and 20% L929 cell supernatant (containing macrophage colony stimulating factor).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To examine mRNA expression, cells were isolated using the miRVana isolation kit (Life Technologies) according to manufacturer specifications.
|
| Label |
biotin
|
| Label protocol |
Samples were enzymatically labeled using BioArray HighYield RNA Transcript Labeling kit (Enzo)
|
| |
|
| Hybridization protocol |
Samples were hybridized to Affymetric 430_2 array using Herring sperm DNA, acetlylated BSA (50 mg/ml) and, Bioarray Eukaryotic Hybridization controls (Enzo Life Sciences), heated 650C 5 minutes, 990C 5 minutes, 450C 5 minutes, centrifuge top speed for 5 minutes, hybridize 16 hours at 450C at 60 rpm; Fluidics script EukGE-WS2v4_450
|
| Scan protocol |
Affymetrix Gene ChIP scanner 30007G using Command Console
|
| Description |
Sample Expr WT2M2
|
| Data processing |
Raw data were normalized with the RMA algorithm implemented in the ‘‘Expression File Creator’’ module from the GenePattern software package. Data were visualized with the Multiplot modules from GenePattern.
|
| |
|
| Submission date |
Jun 05, 2015 |
| Last update date |
Dec 08, 2015 |
| Contact name |
Mireia Guerau |
| E-mail(s) |
mireia.guerau@osumc.edu
|
| Phone |
614 293 4176
|
| Organization name |
The Ohio State University
|
| Department |
HRS-Medical Laboratory Science
|
| Street address |
453 W 10th Ave
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43220 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE69607 |
Novel transcriptome signatures and markers defining murine macrophages at the extremes of the canonical M1 and M2 polarization spectrum |
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