|
| Status |
Public on Oct 23, 2010 |
| Title |
human macrophages treated with saline, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
human macrophages, 2hr treatment with saline
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
cell type: macrophages
treatment: saline
treatment time: 2 hours
day: 9
|
| Treatment protocol |
Macrophages were infected with live P. gingivalis. In additional cultures, purified LPS or FimA from P. gingivalis were added to the cell culture medium. Cells were incubated at 37°C in an atmosphere containing 5% CO2. Cells and supernatants were harvested at indicated times after incubation with live P. gingivalis or its components.
|
| Growth protocol |
Human PBMC were derived by Ficoll-Hypaque density gradient centrifugation from buffy coats of different healthy blood donors obtained from the Interstate Blood Bank (Memphis, TN). CD14-positive monocytes were enriched from PBMC to >95% purity by immunomagnetic elimination of T cells, NK cells, B cells, dendritic cells, and basophils using the Monocyte Isolation Kit II. Purity of monocytes was determined by flow-cytometric analysis of FITC-conjugated anti-CD14 and PE-conjugated anti-biotin. Purified human monocytes were plated at a density of 2 x 10^6 cells/ml in DMEM with 20% FBS, 10% human serum AB, and 50 µg/ml gentamicin in 6-well or 10-cm diameter tissue culture plates for 5 days at 37°C in a humidified atmosphere containing 5% CO2. On days 5 and 7, half of the medium were removed and replaced with medium lacking FBS. Media on the cultured macrophages were replaced with fresh DMEM containing 1% human serum on day 9, 1 hour before experiments were begun.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Two hours after infection with P. gingivalis, or treatment with P. gingivalis LPS, FimA, or saline (control), human macrophages were washed three times with ice cold PBS, and total RNA was extracted using an RNeasy Mini Kit according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
GeneChip Expression Amplification Reagents kit-30 reactions (P/N 900449).
|
| |
|
| Hybridization protocol |
Hybridization was conducted according to the Affymetrix GeneChip Manual. Twenty micrograms of in vitro transcription material were used on each GeneChip Human Genome U133 Plus 2.0 array, which contains 54674 gene probe sets.
|
| Scan protocol |
Affymetrix GeneChIP Scanner 3000 7G.
|
| Description |
Gene expression data from human macrophages treated with saline (control) for 2 hours.
|
| Data processing |
Data were processed using R/Bioconductor software. The raw data were read from CEL files into the object of class AffyBatch. We compared the expression of genes in macrophages under three conditions: live P. gingivalis-stimulated versus control, LPS-stimulated versus control, and FimA-stimulated versus control. For each condition, background correction and quantile normalization were adjusted by Robust Multichip Average (RMA) in the Affy package, followed by non-specific filtering to eliminate the uninformative probe sets whose inconsistent phenotypes appeared in the three replicated arrays. The calculation of fold change and adjusted p-value (false discovery rate, FDR) under different conditions was implemented by the limma package.
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| |
|
| Submission date |
Oct 22, 2010 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Han Hu |
| E-mail(s) |
hh1985@bu.edu
|
| Organization name |
Boston University
|
| Department |
Bioinformatics
|
| Street address |
44 Cummington St
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE24897 |
Expression data from human macrophages treated with Porphyromonas gingivalis and its components |
|
| Relations |
| Reanalyzed by |
GSE86362 |
