|
| Status |
Public on Aug 17, 2016 |
| Title |
BMDM_Unstimulated_rep5 |
| Sample type |
RNA |
| |
|
| Source name |
BMDM Unstimulated
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 8 weeks
tissue: bone marrow
cell type: bone marrow-derived macrophages (BMDMs)
cytokine stimulation: none (unstimulated)
|
| Treatment protocol |
BMDM were isolated after 7 days of CSF-1 treatment. Cells were cultured in suspension in 7 ml Teflon bags (PermaLife) and stimualted for 24 hours with 10 ng/ml of the indicated cytokines in DMEM +10% FBS+ Pen/Strep + CSF-1 10 ng/ml.
|
| Growth protocol |
To derive primary bone marrow-derived macrophages (BMDM), bone marrow was harvested from wild-type C57BL/6 mice aged 8 weeks by flushing femurs and tibias with DMEM media using 25-gauge needles. The marrow was passed through a 40 μm strainer and cultured in FEP (Teflon) cell culture bags (American Fluoroseal or PermaLife) with DMEM media containing 10% FBS, supplemented with 10 ng/mL recombinant mouse CSF-1 (R&D Systems). Bone marrow cells were cultured for 7 days, with fresh CSF-1-containing medium replacing old medium every other day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted, washed with PBS, and resuspended in TriZOL. RNA was isolated following a chloroform extraction, isopropanol precipitation, and ethanol wash.
|
| Label |
Biotin
|
| Label protocol |
75 ng total RNA converted to cRNA using Affymetrix GeneChip®3' IVT Kit and in vitro transcription resulting into biotin-labeled cRNA, fragmented prior to hybridization (Affymetrix Expression Analysis Technical Manual, as described).
|
| |
|
| Hybridization protocol |
15 μg of cDNA was hybridized for 16 hr to the Affymetrix array as described in the Expression Analysis Technical Manual. GeneChips were washed and stained in the Affymetrix Fluidics Station FS450.
|
| Scan protocol |
Affymetrix GeneArray scanner 7G using the GeneChip Command Console version 3.2 as CEL files
|
| Data processing |
Feature intensity was extracted by Affymetrix GCOS 1.4. All analysis was completed in R using the Bioconductor suite of packages. Expression values were computed using the robust multi-array average (RMA) method, and then quantile normalized
|
| |
|
| Submission date |
Jul 08, 2015 |
| Last update date |
Aug 17, 2016 |
| Contact name |
Robert L Bowman |
| E-mail(s) |
bowmanr@mskcc.org
|
| Phone |
6468882059
|
| Organization name |
MSKCC
|
| Department |
HOPP
|
| Lab |
Levine Lab
|
| Street address |
430 East 67th Street
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE70626 |
TH2 cytokines promote tumor progression via regulating cathepsin secretion in tumor-associated macrophages |
|
