|
| Status |
Public on Mar 15, 2013 |
| Title |
Monocyte NS 6h 1 |
| Sample type |
RNA |
| |
|
| Source name |
Monocytes unstimulated (6 hours)
|
| Organism |
Homo sapiens |
| Characteristics |
stimulation: NO
treatment: NS
time: 6h
cell type: Monocyte
ifng: 0
il4: 0
|
| Treatment protocol |
Monocytes and monocyte-derived macrophages were stimulated with 20 ng/ml recombinant human IFN-gamma (PeproTech, Neuilly-sur-Seine, France) or IL-4 (AbCys, Paris, France) for 6 or 18 hours.
|
| Growth protocol |
Leukopacks from normal blood donor buffy coats were provided from the Etablissement Français du Sang (Marseille, France). Peripheral blood mononuclear cells were isolated from blood samples after deposition over a Ficoll density cushion. They were then subjected to CD14+ magnetic cell sorting using a monocyte isolation kit (Miltenyi Biotec, Paris, France). This procedure resulted in more than 95% monocyte purity, as assessed by flow cytometry. CD14+ cells (10e6 cells per assay) were differentiated into macrophages. In brief, they were incubated in RPMI 1640 containing 20 mM HEPES, 10% human serum AB+, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Saint Aubin, France) for 3 days, and fetal calf serum was substituted by human serum for 4 supplementary days. More than 95% of the cells were macrophages, as assessed by CD68 expression and CD14 down-modulation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using an RNeasy Mini kit (Qiagen, Courtaboeuf, France). The quantity and quality of total RNA were assessed using the Nanodrop (Thermo Scientific, Illkirch, France) and a 2100 Bioanalyzer (Agilent Technologies, Massy, France). The RNA was eluted in 30 µl of water and stored at -20°C.
|
| Label |
Cy3
|
| Label protocol |
In brief, 250 ng RNA were labeled with cyanine-3-CTP using the Low RNA Input Fluorescent Amplification Kit from Agilent Technologies.
|
| |
|
| Hybridization protocol |
RNA was analyzed using microarray chips including 45,000 probes (4x44K Whole Human Genome, Agilent Technologies) and One-color Microarray Based Gene Expression Analysis. Hybridization was performed at 65°C using the in situ Hybridization Plus kit (Agilent Technologies) for 17 hours..
|
| Scan protocol |
he arrays were scanned with a pixel size of 5 µm with the DNA Microarray Scanner G2505B. Image analysis and correction of intra-array signals were performed with the Feature Extraction Software A.9.1.3 (Agilent Technologies).
|
| Description |
Monocytes unstimulated (6 hours)
|
| Data processing |
Microarray data analysis was performed using the R and the Bioconductor software suite. Raw data were filtered for probes with signals below background and normalized using the Agi4x44PreProcess library. Unsupervised and supervised analyses were done using hierarchical clustering, principal component analysis (PCA) (made4 library) and Significance Analysis of Microarray (SAM) algorithm (siggenes library). Genes were considered to be differentially expressed when the false discovery rae [23] was below 1% and absolute fold change (FC) was above 2.0 in at least one of the 18 pair combinations.
the "processed signal" from feature extractor was used, with no further background correction
the normalization was done with the "quantile" algorithm as mentioned
the dataset was filtered to remove controls, spot below BG, spot not found (Agilent FLAG), and saturated spots (Agilent FLAG). For each probe, the probe ws removed if the FLAG was set TRUE in > 75% of the samples
the dataset was then summarized for replicated (identical) probes (to keep only one)
|
| |
|
| Submission date |
Mar 15, 2012 |
| Last update date |
Mar 15, 2013 |
| Contact name |
Julien Textoris |
| E-mail(s) |
julien.textoris@gmail.com
|
| Phone |
+33 472 119 546
|
| Organization name |
bioMérieux
|
| Department |
Medical Diagnostic Discovery Department (MD3)
|
| Lab |
Joint Research Unit - bioMérieux / HCL
|
| Street address |
Hôpital Edouard herriot - Pavillon P; 5 place d'Arsonval
|
| City |
Lyon |
| ZIP/Postal code |
69437 |
| Country |
France |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE36537 |
The multifaceted responses of human monocytes to IFN-gamma and IL-4 are not reducible to M1/M2 polarization of Macrophages |
|
