|
| Status |
Public on Aug 06, 2014 |
| Title |
MDM(PBMCcultureadherence)_day5_donor1 |
| Sample type |
RNA |
| |
|
| Source name |
Monocyte-derived macrophages derived from unfractionated PBMC cultures and isolated on day 4 based on adherence and rested 1 day as pure MDM culture
|
| Organism |
Homo sapiens |
| Characteristics |
donor id: donor 1
cell type: Monocyte-derived macrophages (MDM)
purification method: Adherent cells from PBMC culture
days in culture: 5
|
| 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). The adherent cells were considered MDMs after 5 days of differentiation. 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 day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA purification using TRIzol Reagent
|
| Label |
FAM (TaqMan)
|
| Label protocol |
cDNA was generated using the TaqMan Reverse Transcription Kit and Megaplex Primer Pool A v2.0. Preamplification was performed using TaqMan PreAmp Master Mix and Megaplex PreAmp Primer Pool A v2.0. Finally, TLDA cards were loaded with pre-amplified reactions and PCR was performed on a 7900HT Fast Real Time PCR System (Applied Biosystems).
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
MDMs(PBMC) 1
|
| Data processing |
SDS v2.3 and SDS RQ Manager v1.2 software (Applied Biosystems); the non_normalized.txt contains Non-normalized Ct values
|
| |
|
| Submission date |
Aug 05, 2014 |
| Last update date |
Aug 06, 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 |
GPL14851 |
| Series (2) |
| GSE60110 |
Regulation of Activation-Associated MicroRNA Accumulation Rates during Monocyte-to-Macrophage Differentiation |
| GSE60550 |
Regulation of Activation-Associated mRNA and MicroRNA Accumulation Rates during Monocyte-to-Macrophage Differentiation |
|
