cell line: U87-MG cell line passage: 40 duration of cultivation: 72 h type of cultivation plate: Boyden chamber top chamber (single 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 3
Data processing
The data were log2 transformed and normalised using robust spline normalisation with lumi package in R statistical environment.