|
| Status |
Public on Apr 13, 2011 |
| Title |
MDSC_Syngenic_2 |
| Sample type |
RNA |
| |
|
| Source name |
MDSCs
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell type: myeloid-derived suppressor cells (MDSCs)
transplantation: Syngeneic
state: Syngeneic
day post transplantation: 100
|
| Treatment protocol |
Lewis 1W (LEW.1W, haplotype RT1u) and Lewis 1A (LEW.1A, haplotype RT1a) rats (Centre d’Elevage Janvier, Le Genest-Saint-Isle, France) share the same genetic background but are MHC I and II mismatched. Heterotopic transplantations of 8 to 12 weeks old LEW.1W kidneys on LEW.1A animals were carried out according to the technique described by Ono & Lindsey. For syngeneic transplantations LEW.1A kidneys on LEW.1A recipients were achieved. Live-sustaining allotransplantations were performed aseptically and a binephrectomy was performed 7 days after transplantation as previously described. Blood urea and creatinine and urine protein/creatinine ratios were measured throughout the posttransplant period. Blood urea < 8 mM and blood creatinine < 40 mM were considered as normal. Transplant tolerance induction was obtained by treating rats with anti-CD28 (JJ319) antibody.
|
| Growth protocol |
Rats were maintained in an SPF-free animal facility according to the institutional guidelines of the “Institut National de la Santé et de la Recherche Médicale” (INSERM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral blood leucocytes were isolated from heparinized blood by removing erythrocytes with hypotonic lysis solution. A depletion of MHC class II + and CD3+ cells was performed with specific mAbs; followed by anti-mouse IgG-coated Dynabeads (Life Technologies). MDSCs were identified by staining 30 min at 4°C with purified anti-rat CD80/86 (3H5/24F) mAbs and FITC-conjugated anti-mouse IgG secondary Ab (Jackson ImmunoResearch Laboratories). MDSCs were isolated using automated magnetic cell sorting (AutoMACS, Miltenyi) of cells based on their expression of CD80/86. Total RNA from MDSCs was prepared using the TRIzol (Invitrogen Life Technologies) extraction method. Ten micrograms of total RNA was cleaned up using RNeasy columns (Qiagen). RNA quantity and quality were determined using a NanoDrop spectrophotometer and an Agilent 2100 bioanalyzer. A rRNA 28S/18S ratio of 1.0 ± 0.1 was considered as acceptable for RNA amplification.
|
| Label |
Digoxigenin-UTP
|
| Label protocol |
The Applied Biosystems rat genome survey microarray (part no. 4337467) contained 26,857 60-mer oligonucleotide probes representing 27,088 individual rat genes. An additional 3400 control spots were present on the chip to cover various steps in the hybridization process. Digoxigenin-UTP labeled cRNA was generated and amplified from 0.5 µg of total RNA from each sample using an Applied Biosystems NanoAmp chemiluminescent reverse transcriptase-in vitro transcription (RT-IVT) labeling kit (part no. 4365715).
|
| |
|
| Hybridization protocol |
Array hybridization was performed for 16 h at 55°C. An additional 3400 control spots were present on the chip to cover various steps in the hybridization process.
|
| Scan protocol |
Chemiluminescence detection, image acquisition, and analysis were performed using the Applied Biosystems chemiluminescence detection kit (part no. 436875D), analyzer (part no. 4338036) and version 1.1 analyzer software (part no. 4336391) according to the manufacturer’s protocol.
|
| Description |
Syngeneic
antibodies details: Purified anti-MHC class II (OX6) and anti-CD3 (G4.18) were prepared in our laboratory from the corresponding hybridomas obtained from the European Cell Culture Collection (Salisbury, UK). Goat anti-mouse IgG Dynabeads were purchased from Invitrogen/Life Technologies, cat n 110.33. Anti-CD80 (3H5) and anti-CD86 (24F) mouse antibodies were prepared in our laboratory from hybridoma given by Dr. H. Yagita (Juntendo University School of Medicine, Tokyo, Japan). FITC-conjugated anti-mouse was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA), cat n 715-095-150. Anti-FITC Microbeads were from Miltenyi, cat n 130-048-701.
|
| Data processing |
Microarray raw data were analyzed by the R language and environment for statistical computing and graphics. Genes were identified using the Panther Protein Classification System Probe ID database.
|
| |
|
| Submission date |
Apr 12, 2011 |
| Last update date |
Apr 13, 2011 |
| Contact name |
Nahzli DILEK |
| E-mail(s) |
nahzli.dilek@univ-nantes.fr
|
| Phone |
0033240087492
|
| Fax |
0033240087411
|
| Organization name |
INSERM UMR643
|
| Street address |
30 Bd Jean-Monnet, CHU Hôtel-Dieu
|
| City |
NANTES |
| ZIP/Postal code |
44093 |
| Country |
France |
| |
|
| Platform ID |
GPL2996 |
| Series (1) |
| GSE28545 |
Comparison between blood myeloid-derived suppressor cells (MDSCs) extracted from syngeneic kidney transplanted recipient and tolerant kidney allografted recipient induced by anti-CD28 antibody |
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