Exosomes were prepared from the supernatant of MC/9 cells. The cells were harvested, centrifuged at 500 g for 10 min to eliminate cells and at 23 000 g for 20 min, followed by filtration through 0.22 µm filter to remove cell debris. Exosomes were pelleted by ultra-centrifugation at 145 000 g for 70 min.
Growth protocol
MC/9 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0, 05 mM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS and 10% T-Cell Culture Supplement with conA (Rat T-stim) at 37 °C, 5% CO2 in a humidified incubator.
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Hybridisation cocktail was prepared with the biotinylated and fragmented cRNA, control oligonucleotide B2, herring sperm DNA, acetylated BSA, 4-morpholinepropanesulfonic acid (MES), Tween 20, and bacterial cRNA controls respectively. The hybridisation cocktail was heated to 99°C for 5 min and to 45°C for 5 min, and was briefly centrifuged before hybridization. The prewet GeneChipTM probe array cartridge was filled with the hybridisation cocktail and incubated on rotation, 60 rpm at 45°C for 16–18 h. The cartridge was then subjected to an automated washing procedure, using the GeneChipTM Fluidics Station 400 and with a non stringent wash buffer, containing saline, sodium, phosphate, EDTA (SSPE), Tween 20 and antifoam according to the Affymetrix manual. A final wash with the stringent buffer, containing MES-sodium salt and Tween 20 was performed. The probe array was then stained with a solution of acetylated BSA, streptavidin and R-phycoerythrin in stain buffer, containing sodium-MES and Tween 20 and antifoam, followed by non stringent wash buffer. A second stain was performed with a solution of acetylated BSA, normal goat IgG and biotinylated goat antistreptavidin antibody in stain buffer. A third staining step with streptavidin R-phycoerythrin was performed as described above, before a final one with non-stringent wash buffer at 30°C.
Scan protocol
The probe arrays were scanned with the Gene Array Scanner (Affymetrix, Inc. www.affymetrix.com).
Description
Gene expression data from exosomes derived from mast cell line MC/9
Data processing
The software GenChip Operating System 1.2 (GCOS; Affymtrix Inc.) were used for data processing.
