|
| Status |
Public on Feb 23, 2009 |
| Title |
LTB4 stim |
| Sample type |
RNA |
| |
|
| Source name |
macrophage, ex vivio stimulation TB extract for 4 hrs
|
| Organism |
Homo sapiens |
| Characteristics |
5 day monocyte-derived macrophage
Latent
|
| Treatment protocol |
MDMs were stimulated ex-vivo with either 5 µg/ml of whole cell lysate of M. tuberculosis H37Rv (obtained from the Mycobacteria Research laboratories at Colorado State University, USA) or PBS control for 4 hours.
|
| Growth protocol |
Peripheral Blood Mononuclear Cells (PBMCs) were separated from heparinized whole blood by Lymphoprep (Asix-Shield, Norway) gradient centrifugation according to the manufacturer's protocol. 20 ml of blood was slowly added to the tube containing 20ml of Lymphoprep and spun at 2800 rpm for 24 minutes at room temperature with low brake. The PBMC layer was transferred to a new tube and washed 3 times with cold PBS 3% fetal calf serum (FCS) to remove platelets. To derive monocytes, 1-1.5x107 PBMCs were plated in a well of a 6-well plate (Nunc, Denmark) in RPMI-1640 (Sigma, Germany) with 10% heat-inactivated fetal calf serum (FCS; Sigma, Germany), 2mM L-glutamine and 100 units of penicillin for 2 hours at 370C. Non-adhered cells were washed off 3 times with PBS 3 % FCS. The cells were plated in RPMI media for 5 days at 370C 5% CO2 to make monocyte-derived macrophages (MDMs).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells grown in a monolayer were treated with Trizol (Invitrogen, USA) to extract RNA. 1 ml of Trizol was added to a well of a 6-well plate to disrupt cells, dissolve cell components and release RNA. Cell suspension was transferred to a tube and 0.2 ml of chloroform was added to separate the solution into an aqueous phase and an organic phase by centrifugation at 10,000 rpm for 15 minutes. The aqueous phase which contains RNA was transferred to a new tube. Isopropanol was added next with an equal volume to precipitate the RNA by centrifugation at 12,000 for 10 minutes. Then the supernatant was removed and the RNA pellet was washed with 75% ethanol, dried, dissolved in RNase- free water and stored at -80oC.
|
| Label |
biotin
|
| Label protocol |
Standard Affymetrix IVT labeling protocol. See Affymetrix Expression Analysis Technical Manual
|
| |
|
| Hybridization protocol |
Following fragmentation, ten micrograms of adjusted cRNA fromeach sample was hybridized for 16 hours at 45 deg C to Affymetrix GeneChip
|
| Scan protocol |
Scanning was performed using the Affymetrix GeneChip 3000 Scanner. Images were processed into CEL files with the Affymetrix GCOS software.
|
| Description |
Gene expression of monocyte derived macrophages (MDMs) from subjects with three clinical forms of TB including LTB, PTB and TBM (n = 4 in each group) was examined by microarray. MDMs were stimulated either with a whole cell lysate of M. tuberculosis H37Rv or PBS for 4 hours.
|
| Data processing |
Raw CEL intensity data were RMA normalized using R/Bioconductor
|
| |
|
| Submission date |
Apr 08, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Sarah J Dunstan |
| E-mail(s) |
sdunstan@oucru.org
|
| Phone |
84 8 9241761
|
| Fax |
84 8 9238904
|
| Organization name |
1. Oxford University Clinical Research Unit
|
| Department |
Hospital for Tropical Diseases
|
| Street address |
190 Ben Ham Tu, Quan 5
|
| City |
Ho Chi Minh City |
| ZIP/Postal code |
5 |
| Country |
Viet Nam |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE11199 |
Identification of Tuberculosis Susceptibility Genes with Human Macrophage Gene Expression Profiles |
|
| Relations |
| Reanalyzed by |
GSE119087 |
