|
| Status |
Public on Mar 08, 2013 |
| Title |
Nemo-CRE + CLL V132 |
| Sample type |
RNA |
| |
|
| Source name |
BMSC_Nemo wild type_co-cultured with leukemic B-cells
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: Nemo wild type
tissue origin: femora
cell type: bone marrow stromal cells (BMSC)
co-cultured with: CLL V132
|
| Treatment protocol |
After BMSCs had formed a confluent monolayer, primary CLL cells were co-cultured. Typically, 80 x 10x6 primary CLL cells were seeded onto a 10cm dish (56 square cm). After 5 days, CLL cells were separated from co-cultures with stromal cells by repeated washing steps with PBS. Subsequently, single cell suspensions were treated with magnetic anti-CD19 beads to allow removal of remaining CLL cells. Flow-cytometry analysis indicated that more than 99% CD5+CD19+ cells were successfully isolated from CLL-stroma-co-cultures
|
| Growth protocol |
Primary BMSCs were isolated from femora of NemoF/F and Nemoy/F-mice and cultured until they reached 90% confluence. Cells were incubated overnight in a 1:1 mixture of medium (DMEM w/o FCS) and PBS containing 1µM Tat-Cre protein10. Nemo- BMSCs were then cultured at least 8 days after transduction in DMEM supplemented with PS and 10% FCS before co-culture experiments were performed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from BMSCs using the miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer’s instruction.
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the Affymetrix ExpressKit protocol starting from 200 ng total RNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45°C on GeneChip MG-430_2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 using the Affymetrix Hybridization, Wash and Stain Kit.
|
| Scan protocol |
Arrays were scanned in a GeneChip Scanner 3000 with G7 update
|
| Description |
Gene expression data of BMSC isolated from uninduced strain (Nemo-CRE) co-cultured with CLL V132
Nemo-CRE + CLL V132
|
| Data processing |
The data were analyzed with MAS5 using Affymetrix default analysis settings and global scaling to a target intensity of 1000 as normalization method.
|
| |
|
| Submission date |
Mar 10, 2012 |
| Last update date |
Mar 08, 2013 |
| Contact name |
Ludger Klein-Hitpass |
| E-mail(s) |
[email protected]
|
| Phone |
+49 201 723 85552
|
| Organization name |
Institut fuer Zellbiologie
|
| Department |
Universitaetsklinikum
|
| Lab |
BioChip Lab
|
| Street address |
Virchowstr. 173
|
| City |
Essen |
| ZIP/Postal code |
D-45122 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (2) |
| GSE36415 |
Effect of NF-kappaB activation in bone marrow stromal cells co-cultured with CLL cells |
| GSE36416 |
Protein kinase C-beta dependent activation of NF-kB in stromal cells is indispensable for the survival of chronic lymphocytic leukemia in B-cells in vivo. |
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