|
| Status |
Public on Dec 20, 2011 |
| Title |
hMSC4_in-direct_co-culture_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
MSC-4 cell culture
|
| Organism |
Homo sapiens |
| Characteristics |
stem cell clone: MSC-4
cell line passage: 7
duration of cultivation: 72 h
type of cultivation plate: Boyden chamber bottom chamber (co-culture)
|
| Treatment protocol |
Cells were not treated in any way. In co-culture experiments, cells were left to grow in separate chambers of Boyden chambers for 72 h, as described above.
|
| Growth protocol |
Human MSCs derived from bone marrow were purchased from Lonza Bioscience, Walkersville Inc. (MSC-4: 6F4085). After resuscitation cells were plated and grown according to manufacturers recommendations. Briefly, hMSC were grown in Dulbecco medium (DMEM, Sigma 5921,) and 10% fetal bovine serum (FBD, PPL), supplemented with 100 U penicilin, 1000 U streptomycin, 2mM L-Glutamine, Na-Pyruvate and non-essential amino acids. During cultivation (37°C, 5% CO2) medium changes were carried out three times weekly. Cells were passaged after reaching 75% confluence and plated at density 5000 cells/cm2. Human brain tumor cell line (U87-MG) was purchased from the American Type Culture Collection –ATCC (Manassas, VA, USA). Cells were cultured in the same medium as hMSC cells. They were passaged at 75% confluency and seeded at a density 15 000 cells/cm2. All experiments were further performed in a medium containing 10% serum. hMSC of passage 7 and glioma cell lines of passage 40 were used in the experiments. The co-culture experiments were set up in duplicates using 6-well Transwell inserts (Costar, Cornig incorporated, NY) with 0.4 um pore size. On the bottom of the well the hMSC were plated at the density of 105 cells in 2 ml, whereas the U87-MG cells were always plated in the insert at the density of 80 000 cells/mL. Cells were always left to adhere for 3h before being put together for 72 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from three biological replicates of monocultures and indirect co-cultures of MSC and U87-MG cells using Trizol reagent (Invitrogen Limited, Paisley, Scotland UK) following manufacturer’s instructions. RNA was column purified with RNeasy Mini Kit (Qiagen,UK) and integrity confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays.
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol
|
| Scan protocol |
Standard Illumina scanning protocol (BeadStudio).
|
| Description |
biological replicate 1
|
| Data processing |
The data were log2 transformed and normalised using robust spline normalisation with lumi package in R statistical environment.
|
| |
|
| Submission date |
Dec 22, 2010 |
| Last update date |
Dec 20, 2011 |
| Contact name |
Kristina Gruden |
| E-mail(s) |
kristina.gruden@nib.si
|
| Organization name |
National Institute of Biology
|
| Department |
Department of Biotechnology and Systems Biology
|
| Street address |
Vecna pot 111
|
| City |
Ljubljana |
| ZIP/Postal code |
1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL6884 |
| Series (1) |
| GSE26283 |
Human Mesenchymal Stem Cells Exploit the Immune Response Mediating Chemokines to Impact the Phenotype of Glioblastoma |
|
