|
| Status |
Public on Aug 21, 2014 |
| Title |
MDM(PBMCculture+LPS)_day6_donor1 |
| Sample type |
RNA |
| |
|
| Source name |
Human monocytic cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: monocyte
cd14 microbead purification?: No
monocytes co-cultured with nonmonocytic pbmcs?: Yes
number of days cultured in vitro: 6
non-monocytic cells removed on day 5?: Yes
|
| 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 |
Test
|
| 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 |
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