|
| Status |
Public on Dec 30, 2009 |
| Title |
AVZ 27_GMCSF_Serum |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
GM-CSF monocyte-derived macrophage
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: GM-CSF induced human monocyte-derived macrophage
|
| Treatment protocol |
For the isolation of PMBC from Buffy Coat and removal of dead cells, a density gradient centrifugation using Ficoll–Paque (GE Healthcare) gradient was used. PMBC were washed with PBS and the supernatant completely removed. The cell pellet was re-suspended in 80 µl of PBS/BSA buffer per 107 total cells and 10 µl of MACS CD14 (Miltenyi Biotec) were added carefully, mixed and incubated for 15 min at 6°-12°C. The cells were washed by adding 10-20X labelling volume of buffer, centrifuged at 300 g for 10 min. The cells were re-suspended in 500 µl of buffer before magnetic separation as described by the manufacturer.
|
| Growth protocol |
The human monocytes CD14+ obtained after MACS separation were cultured at a concentration of 1x 106 cells/ml in pure RPMI 1640 medium in P6 Petri dishes. After 1h of incubation at 37°C, non adherent cells were removed and the medium was replaced with RPMI 1640 Glu+ medium supplemented with 100U/ml penicillin, 100 µg/ml streptomycin and with 10 % FCS and 10 ng/ml of macrophage colony stimulating-factor (M-CSF, SIGMA) or with 10 % FCS and 10 ng/ml of granulocyte macrophage colony-stimulation factor (GM-CSF, SIGM) or with 10 % human serum only. Cells were incubated for 6 days at 37°C/5% CO2 to induce the phagocytic differentiation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from monocytes derived macrohages using RNeasy mini kit of Qiagen.
|
| Label |
Cy5
|
| Label protocol |
RNA was amplified and labeled with Cy3 or Cy5
|
| |
|
| Channel 2 |
| Source name |
peritoneal macrophage
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: peritoneal macrophage
|
| Treatment protocol |
For the isolation of PMBC from Buffy Coat and removal of dead cells, a density gradient centrifugation using Ficoll–Paque (GE Healthcare) gradient was used. PMBC were washed with PBS and the supernatant completely removed. The cell pellet was re-suspended in 80 µl of PBS/BSA buffer per 107 total cells and 10 µl of MACS CD14 (Miltenyi Biotec) were added carefully, mixed and incubated for 15 min at 6°-12°C. The cells were washed by adding 10-20X labelling volume of buffer, centrifuged at 300 g for 10 min. The cells were re-suspended in 500 µl of buffer before magnetic separation as described by the manufacturer.
|
| Growth protocol |
The human monocytes CD14+ obtained after MACS separation were cultured at a concentration of 1x 106 cells/ml in pure RPMI 1640 medium in P6 Petri dishes. After 1h of incubation at 37°C, non adherent cells were removed and the medium was replaced with RPMI 1640 Glu+ medium supplemented with 100U/ml penicillin, 100 µg/ml streptomycin and with 10 % FCS and 10 ng/ml of macrophage colony stimulating-factor (M-CSF, SIGMA) or with 10 % FCS and 10 ng/ml of granulocyte macrophage colony-stimulation factor (GM-CSF, SIGM) or with 10 % human serum only. Cells were incubated for 6 days at 37°C/5% CO2 to induce the phagocytic differentiation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from monocytes derived macrohages using RNeasy mini kit of Qiagen.
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified and labeled with Cy3 or Cy5
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed using the Agilent in situ hybridization kit. For each sample, 750 ng of Cy5-labeled, linearly amplified cRNA was mixed with equal amounts of Cy3-labelled, linearly amplified reference cRNA. The mixture was then fragmented by incubation with the fragmentation buffer at 60°C for 30 minutes. An equal amount of 2x hybridization buffer was added to the fragmented cRNA mixture and hybridized to whole-genome oligo arrays (Human-25K, Réseau National des Génopoles, France) at 60°C for 17 hours
|
| Scan protocol |
Fluorescent images of hybridized microarrays were obtained with a GenePix 4000 Axon Instruments scanner and provessed with the GenePix software (Axon Instruments).
|
| Description |
n/a
|
| Data processing |
The Bioconductor packages marray and Limma were used to perform quality control and preprocessing. Background correction method: normexp Within array print-tip loess normalization was performed for each spot followed by between array quantile normalization. bad spots were filtered out. Log2 ch1/ch2.
|
| |
|
| Submission date |
Sep 25, 2009 |
| Last update date |
Sep 25, 2009 |
| Contact name |
Seraya Maouche |
| E-mail(s) |
[email protected]
|
| Fax |
+33140779728
|
| URL |
http://genecanvas.idf.inserm.fr/news.php
|
| Organization name |
INSERM U937
|
| Department |
Cardiovascular Genomics
|
| Street address |
91 Bd de l'Hôpital
|
| City |
Paris |
| State/province |
Paris |
| ZIP/Postal code |
75634 Paris Cedex 13 |
| Country |
France |
| |
|
| Platform ID |
GPL1946 |
| Series (1) |
| GSE18275 |
Macrophages heterogeneity in human atherosclerotic plaques |
|
