|
| Status |
Public on Mar 20, 2008 |
| Title |
Anti-Trem1/LPS 2 hrs Donor 6 |
| Sample type |
RNA |
| |
|
| Source name |
Monocytes__Anti-Trem1/LPS_2_hrs
|
| Organism |
Homo sapiens |
| Characteristics |
Anonymous Donor
|
| Growth protocol |
Human monocytes were purified from anonymous healthy donor Buffycoats obtained from Massachusetts General Hospital. Buffycoats were stored at 4°C overnight for cell isolation the following day. Monocytes were isolated by negative selection using RosetteSep (Stem Cell Technologies, 15068) according to the manufacturer’s protocol by density centrifugation over Histopaque (SIGMA, H8889). Monocytes were maintained in RPMI 1640 (Mediatech, Inc., 15-040-CV) supplemented with 10% fetal bovine serum (SIGMA, F-9423) that had been heat-inactivated. All incubations were at 37°C in a tissue culture incubator maintained at 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated after 2 hour treatment using QIAshredders and RNeasy miniprep kits according to the manufacturer’s protocol (QIAGEN, 79654 and 74104, respectively). Total RNA yields ranged from 1-6 mg. Residual genomic DNA was removed by DNase treatment and phenol/chloroform extraction followed by ethanol precipitation using standard techniques.
|
| Label |
Biotin
|
| Label protocol |
Microarray on Affymetrix Human Genome U133_plus 2.0 arrays was performed according to established protocols
|
| |
|
| Hybridization protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| Scan protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Description |
Sample_Treatment_[C] = Anti-Trem1
Sample_Treatment_amount_[C] = 5 ug/ml
Sample_Treatment_2_[C] = Lipopolysaccharide
Sample_Treatment_2_amount_[C] = 1 ng/ml
Sample_Treatment_Source = R&D Systems Cat MAB1278
Sample_Treatment_2_Source = Sigma Cat L2262
Sample_Treatment_protocol = For TREM-1 activation, tissue culture treated plates were pre-incubated with an appropriate volume of 5 mg/ml a-TREM-1 antibody (R&D Systems, MAB1278) in PBS overnight in a tissue culture incubator. Wells were washed twice with PBS immediately prior to cell addition. As a control, wells received the same treatment with an isotype-matched murine IgG1 antibody (a-E.tenella; Wyeth). For LPS treatment, gel filtration chromatography purified LPS from Salmonella enterica (SIGMA, L2262) was added to a final concentration of 1 ng/ml. In the wortmannin studies, cells in RPMI/10% FBS were pre-incubated with wortmannin (SIGMA, W1628) at a final concentration of 100 nM for 30 minutes in polypropylene tubes prior to seeding into tissue culture wells. The final DMSO concentration in control and wortmannin treated cultures was 0.1%.
|
| Data processing |
GeneChips were scanned using the Affymetrix 3000 Scanner
|
| |
|
| Submission date |
Dec 20, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Scott Jelinsky |
| E-mail(s) |
Scott.Jelinsky@pfizer.com
|
| Phone |
617-674-7272
|
| Organization name |
Pfizer
|
| Department |
Inflammation and Immunology
|
| Lab |
Computational Precision Medicine
|
| Street address |
610 Main Street
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE9988 |
Innate immune repsonses to TREM-1 activation |
|
| Relations |
| Reanalyzed by |
GSE119087 |
