|
| Status |
Public on Mar 01, 2009 |
| Title |
1_NK Kit+ |
| Sample type |
RNA |
| |
|
| Source name |
NK cell subsets from C57Bl/6 mice
|
| Organism |
Mus musculus |
| Characteristics |
CD3-NK1.1+CD117+ NK cells compared to CD3-NK1.1+CD117- NK cells from spleen. Strain: C57Bl/6. Gender: female. Age: 7 weeks. C57Bl/6 mice were obtained from C.River Laboratories (L’Arbresle, France) and the Centre d’Elevage Janvier (Le Genest-st-Isle, France).
|
| Biomaterial provider |
Material provided from laboratory of Prof. Laurence Zitvogel, Immunology, INSERM U805, Instiut Gustave Roussy, Villejuif, France.
|
| Treatment protocol |
Two subpopulations of NK cells are sorted from mouse spleens with a FACS Vantage instrument (BD Bioscience). NK kit+ are defined as CD3-NK1.1+CD117+ cells and NK kit- as CD3-NK1.1+CD117- cells. Splenocytes from C57Bl/6 mice were sorted in two steps. First, they were enriched in NK cells (NK1.1+/CD3-) by a magnetic separation using NK cell Isolation Kit (Miltenyi Biotec). Second, enriched NK cells were stained with FITC-conjugated anti-CD117 (c-kit) and PE-conjugated anti-NK1.1 monoclonal antibodies to allow a sorting of NK kit+ and NK kit- cells. Antibodies were purchased from Pharmingen, San Diego, CA or eBioscience, San Diego, CA. The purity of NK cells was around 98% after cell sorting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
NK kit+ and NK kit- subsets are stocked at -80°C in Trizol and then used for isolation of RNA. RNA isolation was performed according the manufacturer´s protocol. Total RNA was subsequently purified with Qiagen MinElute Cleanup Kit according to the manufacturer´s protocol.
|
| Label |
biotin
|
| Label protocol |
Biotin-labeled cRNA was synthesized according to the instruction manual of the Illumina® TotalPrep™ RNA Amplification Kit (Applied Biosystems, Darmstadt, Germany) using 50 – 100 ng total RNA.
|
| |
|
| Hybridization protocol |
1.5 µg cRNA was hybridized for 16 h to Sentrix Mouse-6 Expression BeadChips (Illumina) according to the Sentrix Beadchip hybridization protocol of the Illumina Beadstation 500X System manual.
|
| Scan protocol |
Samples were scanned using Illumina´s Direct-Hyb-Protocol on Illumina Beadstation 500X.
|
| Description |
NK-Kit+ cells
|
| Data processing |
Raw data were extracted using Illumina Beadstudio Software V 3.1.1.0
Quantile normalization was performed using the IlluminaGUI R-package
(http://IlluminaGUI.dnsalias.org).
|
| |
|
| Submission date |
Oct 22, 2007 |
| Last update date |
Dec 21, 2011 |
| Contact name |
Joachim L Schultze |
| E-mail(s) |
j.schultze@uni-bonn.de
|
| Organization name |
University of Bonn
|
| Department |
LIMES Institute
|
| Lab |
Genomics & Immunoregulation
|
| Street address |
Carl-Troll-Str. 31
|
| City |
Bonn |
| ZIP/Postal code |
53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4234 |
| Series (1) |
| GSE9431 |
Regulatory NK cells: IL-18-elicited NK cells with immunosuppressive functions |
|
