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Sample GSM211506 Query DataSets for GSM211506
Status Public on Sep 15, 2007
Title IL-1, Rep2
Sample type RNA
 
Source name Interleukin 1 replicate 2
Organism Homo sapiens
Characteristics Human monocyte-derived macrophages obtained from peripheral blood mononuclear cells
Treatment protocol Just before the experiment with cytokines the medium was changed to DMEM containing 0.5% of pooled human serum and the cells were stimulated with IL-1 (15 ng/ml), or IL-6 (25 ng/ml) for the period of 4 h. These rather low concentrations of cytokines that may occur in vivo were selected in preliminary trials as sufficient to stimulate the expression of manganese superoxide dismutase (MnSOD).
Growth protocol Human monocyte-derived macrophages (hMDMs) were obtained from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human blood of 7 healthy donors using a Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient. Cells from each donor were split into three aliquots to stimulate them later with a mock stimulant, IL-1 and IL-6 and plated at 4x107 in PrimariaTM 25cm2 cell culture flasks with vented caps (Becton Dickinson, Franklin Lakes, NJ, USA) in RPMI1640 (Invitrogen) supplemented with 2mM L-glutamine, 50 g/ml gentamycin (Sigma), and 10% pooled heat-inactivated human AB serum. After 24 h, non-adherent PBMCs were removed by washing with complete medium, and adherent cells were cultured in this medium for 7 days with fresh medium changed every 2 days. The MDMs phenotype was controlled, after non-enzymatic detachment of cells, by immunofluorescent staining of CD14 (clone: TÜK4, DakoCytomation Denmark A/S, Glostrup, Denmark), CD16 (clone: DJ130c, DakoCytomation), CD11b (clone: ICRF44, Becton Dickinson and Co., Franklin Lakes, USA), and CD209 (clone: DCN46, Becton Dickinson) and subsequent flow cytometry analysis. The routine procedure used in our laboratory yields cells positive in at least 90% for the first three markers and less than 1% for CD209. It should be added that the adherent cells acquire typical macrophage morphology and extensive phagocytic activity against live Staphylococcus aureus and apoptotic neutrophils. Resting (non-stimulated) cells do not produce proinflammatory cytokines: IL-1, TNF-a or IL-6.
Extracted molecule total RNA
Extraction protocol Total cellular RNA was isolated from unstimulated (control) and cytokine-stimulated cells by Chomczynski and Sacchi procedure.
Label biotin
Label protocol Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
 
Hybridization protocol Following labeling, samples were hybridized to the GeneChip Test3 array (Affymetrix, Santa Clara, CA) for quality control. Fragmented cRNA (15 μg) was used for hybridization to the GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). Arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidic Station 400 (Affymetrix, Santa Clara, CA) according to standard protocol of the manufacturer.
Scan protocol The arrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
Description Interleukin 1 replicate 2
Data processing Data was normalized by dChip 2006 (MBEI)
 
Submission date Jul 18, 2007
Last update date Sep 01, 2016
Contact name Marcin Piechota
E-mail(s) marpiech@if-pan.krakow.pl
Phone (+4812) 6623328
Organization name Institute of Pharmacology PAS
Department Molecular Neuropharmacology
Street address Smetna 12
City Krakow
ZIP/Postal code 31-343
Country Poland
 
Platform ID GPL96
Series (1)
GSE8515 Identification of IL-1 and IL-6-responsive genes in human monocyte-derived macrophages
Relations
Reanalyzed by GSE86363

Data table header descriptions
ID_REF
VALUE MBEI PM/MM signal intensity
ABS_CALL

Data table
ID_REF VALUE ABS_CALL
AFFX-BioB-5_at 60.266 P
AFFX-BioB-M_at 81.465 P
AFFX-BioB-3_at 101.575 P
AFFX-BioC-5_at 226.963 P
AFFX-BioC-3_at 140.56 P
AFFX-BioDn-5_at 307.625 P
AFFX-BioDn-3_at 1076.836 P
AFFX-CreX-5_at 2042.679 P
AFFX-CreX-3_at 2568.122 P
AFFX-DapX-5_at 1195.261 P
AFFX-DapX-M_at 2231.569 P
AFFX-DapX-3_at 1639.176 P
AFFX-LysX-5_at 4813.299 P
AFFX-LysX-M_at 6347.537 P
AFFX-LysX-3_at 6025.515 P
AFFX-PheX-5_at 3267.753 P
AFFX-PheX-M_at 3859.374 P
AFFX-PheX-3_at 4155.945 P
AFFX-ThrX-5_at 4929.996 P
AFFX-ThrX-M_at 5333.7 P

Total number of rows: 22283

Table truncated, full table size 432 Kbytes.




Supplementary file Size Download File type/resource
GSM211506.CEL.gz 2.1 Mb (ftp)(http) CEL
Processed data included within Sample table

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