|
| Status |
Public on Aug 26, 2014 |
| Title |
f-MDSC, before contact, replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
UCB derided CD33+IL4Rα+ sorted and incubated for 4 days with GM-CSF and G-CSF
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: Before contact
cell state: Preactivated cells
cell type: UCB derided CD33+IL4Rα+ sorted and incubated for 4 days with GM-CSF and G-CSF
|
| Treatment protocol |
Sterilely sorted CD33 and IL-4Rα positive cells before and after been co-culture with autologous CD3+ T cells were lysed with TRIZOL and stored at -80◦C till RNA extraction was performed. TRIZOL samples preservation was also used for the control cells line HEK-293.
|
| Growth protocol |
Human Umbilical cord blood (UCB) samples underwent Ficoll-Paque gradient separation and ACK red cell lysis, and then were cultured for 4 days in the presence of GM-CSF and G-CSF (40ng/ml). CD33 and IL-4Rα positive cells were sterilely sorted, part of the cells stored for RNA extraction, and part co-cultured for 3 days with autologous PHA activated CFSE labeled CD3+ T cells either in cell to cell contact (CONTACT) or using a transwell device to avoid cell contact (TRANSWELL). HEK-293 cells were maintained in complete RPMI-140 10% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the miRNA easy kit from Qiagen and following the manufacture instruction.
|
| Label |
biotin
|
| Label protocol |
Affymetrix GeneChip WT Terminal Labeling Kit - sense-strand cDNA is fragmented with a combination of uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 and its yield is further labeled by terminal deoxynucleotidyl transferase (TdT) with the Affymetrix® proprietary DNA Labeling Reagent that is covalently linked to biotin.
|
| |
|
| Hybridization protocol |
GeneChip® Hybridization, Wash, and Stain Kit - Following labeling, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Gene 2.0 ST arrays. GeneChips were washed and stained (streptavidin,R-phycoerythrin conjugate and biotinylated anti-streptavidin) in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
Standard Affymetrix scanning procedures on a GeneChip® Scanner 3000 7G . Affymetrix GeneChip® Command Console® Software (AGCC)- used to view CEL file images.
|
| Description |
Gene expression data of CD33+IL4Ra+ sorted cell before contact with autologus PHA activated CD3+ T cells
|
| Data processing |
RMA of .CEL files using affy Bioconductor library. In RMA, PM values have been background adjusted, normalized using quantile normalization, and expression measure calculated using median polish summarization
|
| |
|
| Submission date |
Nov 14, 2013 |
| Last update date |
Aug 26, 2014 |
| Contact name |
Silvio Bicciato |
| E-mail(s) |
silvio.bicciato@unipd.it
|
| Phone |
+39-049-827-6108
|
| Organization name |
University of Padova
|
| Department |
Molecular Medicine
|
| Street address |
via U. Bassi 59/b
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL17930 |
| Series (1) |
| GSE52376 |
Molecular and cellular characterization of Cord Blood derived, IDO expressing human fibrocystic Myeloid Derived Suppressor Cells |
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