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Sample GSM456459 Query DataSets for GSM456459
Status Public on Dec 30, 2009
Title AVZ 46_MCSF_GMCSF
Sample type RNA
 
Channel 1
Source name M-CSF monocyte-derived macrophage
Organism Homo sapiens
Characteristics cell type: M-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 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 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) seraya.maouche@upmc.fr
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

Data table header descriptions
ID_REF
VALUE normalized log2 ch1/ch2

Data table
ID_REF VALUE
11565 0.255535542
2246 0.074577473
20063 -0.203006479
19538 0.098999796
21822 0.092359952
15637 -0.200449576
16806 -0.039467005
11925 -0.591168302
21856 -0.037771015
2297 -0.022831761
24039 -0.255098766
16319 -0.039647065
133 0.20163329
10046 -0.038866513
1342 -0.020678874
794 0.022375405
18829 -0.376471006
25769 -0.080137204
12454
10612 0.005145997

Total number of rows: 21667

Table truncated, full table size 371 Kbytes.




Supplementary file Size Download File type/resource
GSM456459_AVZ_46_MCSF_GMCSF.gpr.gz 2.0 Mb (ftp)(http) GPR
Raw data provided as supplementary file
Processed data included within Sample table

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