|
| Status |
Public on Feb 27, 2014 |
| Title |
Activated T-Cell Exosomes EA-2 |
| Sample type |
RNA |
| |
|
| Source name |
Activated Exosomes culture #2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Activated T-Cell Exosomes EA-2
origin: Exosome
state: Activated
|
| Treatment protocol |
When indicated, T lymphoblasts were activated with PMA (50ng ml-1) plus ionomycin (500ng ml-1). Exosomes were isolated from cell supernatants by several centrifugation and filtration steps . Briefly, cells were centrifuged (320g for 5 min) and the supernatant filtered through 0.22 μm membranes. Exosomes were pelleted by ultracentrifugation at 100000g for 60 min at 4ºC (Beckman Coulter Optima L-100 XP).
|
| Growth protocol |
Human peripheral blood mononuclear cells were isolated from buffy coats from healthy donors as previously described (Mittelbrunn et al., Nat Commun, 2011) and cultured in RPMI (Sigma) containing 10% fetal bovine serum (FBS; Invitrogen) depleted of bovine exosomes by overnight centrifugation at 100000g)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol reagent (Invitrogen) and the miRNeasy® mini kit (Quiagen),
|
| Label |
Cy3
|
| Label protocol |
miRNA Labeling Kit (Agilent Technologies) was used to label RNA. Basically, 100 ng of total RNA were dephosphorylated and Cyanine 3-pCp molecule was ligated to the 3´ end of each RNA molecule by using T4 RNA ligase.
|
| |
|
| Hybridization protocol |
100 ng of Cy3 labelled RNA were hybridized for 20 hours at 55ºC in a hybridization oven (G2545A, Agilent) set to 15 rpm in a final concentration of 1X GE Blocking Agent and 1X Hi-RPM Hybridization Buffer, according to manufacturer's instructions (miRNA Microarray System Protocol, Agilent Technologies).
|
| Scan protocol |
Arrays were scanned at 5mm resolution on an Agilent DNA Microarray Scanner (G2565BA, Agilent Technologies) using the default settings for mRNA Microarray.Images provided by the scanner were analyzed using Agilent´s software Feature Extraction version 10.7.3.1 and protocol GE1_107_Sep09
|
| Description |
Expression of mRNAs from Exosomes from Activated T-Cells
|
| Data processing |
Data were truncated to 1 and quantiles normalized using GeneSpring Software.Limma package from Bioconductor was used for statistical analysis
|
| |
|
| Submission date |
Sep 18, 2013 |
| Last update date |
Feb 27, 2014 |
| Contact name |
Fatima Sanchez-Cabo |
| E-mail(s) |
fscabo@cnic.es
|
| Phone |
+34 91 4531200
|
| Organization name |
CNIC
|
| Street address |
Melchor Fernandez Almagro
|
| City |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE50971 |
mRNA profiles of activated and resting T cells and their exosomes |
| GSE50972 |
mRNA and miRNA profiles of activated and resting T cells and their exosomes |
|
