|
| Status |
Public on Feb 03, 2011 |
| Title |
CD8 T-Cell Costimulatory blockade treated #2 |
| Sample type |
RNA |
| |
|
| Source name |
CD8 T-cells isolated from the MLR with anti-LFA-1, CTLA4Ig, anti-CD40-ligand antibodies added to the media
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: CD8 T-cells
agent: leuckocyte costimulatory blocking antibodies anti-LFA-1, CTLA4Ig, anti-CD40-ligand
|
| Treatment protocol |
C57BL/6 responder splenocytes were incubated for 5 days with or without 50 µg CTLA4-Ig, 50 µg anti-LFA-1, and 50 µg anti-CD40L.
|
| Growth protocol |
Splenocytes were harvested from either FVB/N (stimulator) or C57BL/6 (responder) mice (Jackson Laboratories) and processed in PBS supplemented with 2% FCS (Invitrogen), n = 4 mice per group. After ACK red blood cell lysis of single cell suspensions, cells were placed in cRPMI media with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen), and 5 µg/mL 2-mercaptoethanol (Sigma-Aldrich). Responder cells were labeled with 5 µM CFSE from CellTrace™ CFSE Cell Proliferation Kit (Invitrogen). MLR was conducted by placing 8x105 FVB/N splenocytes, which had been irradiated with 30 Gy, into each well of a 96-well plate together with 2x105 CFSE-labeled C57BL/6 cells in a total volume of 250 μL cRPMI. Proliferation of C57BL/6 responders was assessed by FACS analysis after 5 days of incubation with or without 50 µg CTLA4-Ig, 50 µg anti-LFA-1, and 50 µg anti-CD40L. Assays were performed in triplicate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following the MLR, responder splenocytes were washed, resuspended in FACS buffer, and incubated with mouse-anti-human CD4+ and CD8+ antibodies (BD Biosciences) for 30 min at 4°C. Responder T-cell subsets were then isolated by FACS on a Vantage SE cell sorter (Becton Dickinson Immunocytometry Systems) analyzing for cells which were double positive for FITC (CFSE) and either CD4 or CD8. Sorted cells were collected and 3x105 cells were counted using a hemocytometer, and total RNA was immediately extracted using the RNeasy micro kit (Qiagen) per manufacturer’s instructions.
|
| Label |
Biotin
|
| Label protocol |
aRNA was prepared according to the protocol for GeneChip 3 IVT Express Kit, Revision 7.
|
| |
|
| Hybridization protocol |
Following fragmentation, aRNA was hybridized on GeneChip Mouse Genome 430 2.0 Array in a GeneChip Hybridization Oven 640. Whshing and Labeled by using GeneChip Fluidicas Station 450.
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000 7G.
|
| Description |
Gene expression data from CD8 T-cells isolated from the MLR with anti-LFA-1, CTLA4Ig, anti-CD40-ligand antibodies added to the media
|
| Data processing |
The data were analyzed with AGCC (Affymetrix Genechip Command Console). Gene-level signal estimates were derived from the CEL files using GeneSpring GX 10.0 software. Summarization of gene expression data was performed by implementing the robust multichip averaging algorithm, with subsequent baseline normalization of the log-summarized values for each probe set to that of the median log summarized value for the same probe set in the control group. Expression data were then filtered to remove probe sets for which the signal intensities for all the treatment groups were in the lowest 20 percentile of all intensity values.
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| |
|
| Submission date |
Jan 18, 2011 |
| Last update date |
Feb 03, 2011 |
| Contact name |
Kitchener D. Wilson |
| E-mail(s) |
kitchwilson@stanford.edu
|
| Organization name |
Stanford University
|
| Street address |
S140 Grant Bldg
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE26669 |
Expression data of alloantigen stimulated splenocytes treated with leukocyte costimulatory blockade antibodies or no treatment |
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