|
| Status |
Public on May 29, 2010 |
| Title |
Unstimulated nTreg cells, replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Unstimulated Treg cells sorted from the the PBMC of a healthy human sample, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: nTreg
|
| Treatment protocol |
PBMCs isolated from random healthy blood donors were first labeled with a CD25-PE antibody, and then isolated with goat anti-mouse labeled magnetic beads by magnetic column separation. Staining of the isolated populations with Foxp3 specific antibody revealed that roughly 50% of the positively selected cells expressed Foxp3, and these cells correlated with the CD25hi population. The percent purity of CD25 isolated cells varied from 50% to 95%. The CD25 selected cells could be sorted based on CD25hi expression. Staining of pre- and post-sorted populations with Foxp3 antibodies showed an increase in purity of TR cells from approximately 60% to over 90% following cell sorting. Sorted cells were able to inhibit the proliferation of autologous CD4 cells in co-culture assays.
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were collected using vacutainers with ACD solution B of trisodium citrate and isolated using Ficoll-Hypaque density centrifugation according to the recommended protocol (Amersham Pharmacia, Uppsala, Sweden).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Purified total RNA (~50ng) was amplified using the two-cycle cDNA synthesis kit (Affymetrix 900432) and cRNA was synthesized, labeled, fragmented and hybridized to the arrays in accordance to standard Affymetrix protocols (Affymetrix, Santa Clara, CA)
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000.
|
| Description |
Gene expression interrogating 54675 transcripts
|
| Data processing |
For both Treg and naive CD4 experiments, two sets of arrays were performed, and the results were averaged. The subset of probe sets whose expression in Tregs increased or decreased by twofold or more relative to naive CD4 cells as a common standard was identified and used for further analysis. The data was normalized using the justRMA algorithm from the Bioconductor suite. Results represent mean fold change values derived from 2 independent arrays for each cell type and scored P < 0.05 by the t-test.
|
| |
|
| Submission date |
May 28, 2010 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Martin Hessner |
| E-mail(s) |
mhessner@mcw.edu
|
| Organization name |
Medical College of Wisconsin
|
| Department |
Pediatrics
|
| Lab |
Max McGee National Research Center for Juvenile Diabetes
|
| Street address |
8701 Watertown Plank Road
|
| City |
Milwaukee |
| State/province |
WI |
| ZIP/Postal code |
53226 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE22045 |
Gene expression profiling of human unstimulated regulatory T cells (Tregs) and naïve CD4+ T cells |
|
| Relations |
| Reanalyzed by |
GSE49910 |
| Reanalyzed by |
GSE86362 |
