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Sample GSM1482178 Query DataSets for GSM1482178
Status Public on Aug 21, 2014
Title Monocyte_day0_donor4
Sample type RNA
 
Source name Human monocytic cells
Organism Homo sapiens
Characteristics cell type: monocyte
cd14 microbead purification?: Yes
monocytes co-cultured with nonmonocytic pbmcs?: No
number of days cultured in vitro: 0
non-monocytic cells removed on day 5?: No
Treatment protocol Where indicated as LPS treated, cells were stimulated with LPS (10ng/ml) on day 5 and samples were collected on day 6.
Growth protocol Monocytic cells were isolated from blood samples using two standard protocols. Each method started with peripheral blood mononuclear cells (PBMCs) isolated using Ficoll-Paque Plus (GE Healthcare) density gradients. In the first method, monocytes were isolated on the day of blood donation directly from PBMCs using CD14 MicroBeads (Miltenyi Biotec). Monocyte samples were collected at the time of sorting to provide a reference/control sample. The remaining purified monocytes were then cultured in tissue culture plates at 1e6 cells/ml in RP-10 supplemented with M-CSF (50 ng/ml). Adherent monocytic cells lysates were collected after incubation for the indicated number of days. The second method, selects MDMs from total PBMC cultures based on adherence. Briefly, PBMCs were cultured in Petri dishes at a density of 5e6 cells/ml in RP-10 (RPMI 1640 (Gibco) with FBS (10% v/v; Gibco) and L-glutamine (2mM; Gibco)) supplemented with M-CSF (5ng/ml; eBioscience). After 4 days of culture, Petri plates were washed extensively with HBSS lacking divalent cations (Gibco) to remove nonadherent cells. The adherent MDMs were trypsinized and pelleted. Upon resuspension at 1e6 cells/ml in RP-10 with M-CSF (5ng/ml), the MDMs were incubated one additional.
Extracted molecule total RNA
Extraction protocol Total RNA purified using TRIzol Reagent
Label FAM (TaqMan Assay)
Label protocol Integrated fluidic circuit (IFC)-based RT-qPCR was performed using the Bio-Mark System for Genetic Analysis (Fluidigm). First, RNA was reverse transcribed using random hexamer-primer reactions with SuperScript III RT. Next, pre-amplification of the RT product was performed using a pool of 48 Individual TaqMan Gene Expression Assays with TaqMan PreAmp MasterMix according to recommendations by Fluidigm. Finally, gene expression was monitored by real time PCR on 48.48 Dynamic Array IFCs.
 
Hybridization protocol n/a
Scan protocol n/a
Description Control
Data processing Fluidigm Real-Time PCR Analysis software version 4.1.2
Non-normalized = Ct; Normalized (in data table and on series record) = Target Ct - Endogenous Control (TBP) Ct; The Fold Change data file contains 2^-deltadeltaCt values for the indicated sample relative to the control sample for each donor and TBP was used as the housekeeping gene.
 
Submission date Aug 20, 2014
Last update date Aug 21, 2014
Contact name Joel Graff
E-mail(s) joel-graff@uiowa.edu
Organization name University of Iowa
Department Internal Medicine
Street address 400 EMRB
City Iowa City
State/province IA
ZIP/Postal code 52242
Country USA
 
Platform ID GPL19055
Series (2)
GSE60549 Regulation of Activation-Associated Transcripts Accumulation Rates during Monocyte-to-Macrophage Differentiation
GSE60550 Regulation of Activation-Associated mRNA and MicroRNA Accumulation Rates during Monocyte-to-Macrophage Differentiation

Data table header descriptions
ID_REF
VALUE Target Ct - Control (TBP) Ct

Data table
ID_REF VALUE
ADORA3 null
CCL1 null
CCL2 -6.301097106
CCL3 -2.675952907
CCL5 -1.243851606
CCL13 null
CCL17 null
CCL18 null
CCL19 null
CCL20 null
CCL22 -4.037189892
CCL26 null
CD163 0.690686814
CLEC10A 1.11550199
COCH null
CXCL5 -3.764503409
CXCL9 null
CXCL10 null
DEFB1 null
F13A1 1.4419057

Total number of rows: 48

Table truncated, full table size <1 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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