cell line: mast cell line HMC-1 total rna source: exosome
Treatment protocol
Growth under normal conditions
Growth protocol
Cell culture and exosome release The human mast cell line HMC-1 (Dr Joseph Butterfield, Mayo Clinic, USA), was cultured in IMDM containing 10% depleted fetal bovine serum (FBS), 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM L-glutamine and 1.2 mM alpha-thioglycerol (all from Sigma-Aldrich). To eliminate exosomes present in serum, FBS was ultracentrifuged at 120 000 g for 90 min using a Ti70 rotor (Beckman optima LE-80k Ultracentrifuge). Peripheral blood mononuclear cells (PBMC) were prepared from peripheral blood of healthy subjects by Ficoll-Plaque density separation. The CD34 cells were obtained from the PBMC by positive isolation using magnetic separation according to the manufacturer’s instruction (Miltenyi Biotec, Germany). CD34 cells were cultured in IMDM (Sigma-Aldrich) containing 10% FBS, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM L-glutamine and 1mM sodium pyruvate. The purity of the separations ranged between 60-85%, as analyzed by flow cytometry using antibody against CD34 (BD Biosciences) in combination with 7-AAD for viability. Exosomes were isolated according to Valadi et al., 2007. Briefly, cells were stimulated with 1 µM calcium ionophore (Sigma-Aldrich) for 30 min, centrifuged at 500 g for 10 min to eliminate cells and at 16 500 g for 20 min, followed by filtration through 0.22 µm filter to remove cell debris. Exosomes were pelleted by ultracentrifugation (Beckman Ti70 rotor) at 120 000 g for 70 min. For PKH67 transfer experiments, exosomes were washed once in a large volume PBS. For electron microscopy, the exosome pellet was diluted in a large volume PBS, filtrated through 0.1 µm filters and pelleted by ultracentrifugation. Exosomes were measured for their protein content using BCA™ Protein Assay Kit (Pierce). Cell viability was assessed using trypan blue exclusion or 7-AAD.
Extracted molecule
total RNA
Extraction protocol
Extraction of total RNA was performed using Trizol according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2001, Affymetrix). The experiment was performed by SweGene (http://www.lth.se/index.php?id=7789) according to the Affymetrix guidelines.
Hybridization protocol
The experiment was performed by SweGene (http://www.lth.se/index.php?id=7789) according to Affymetrix guidelines. Briefly, a hybridisation cocktail was prepared with the biotinylated and fragmented cRNA, control oligonucleotide, 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 pre-wet 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) and controlled by the software GenChip Operating System 1.2 (GCOS; Affymtrix Inc.).
Description
p0739_E1.CEL
Data processing
The expression level Signals were scaled in GCOS 1.2 to give a median array intensity of 100. This was done to enable different arrays to be compared. The program Spotfire DecisionSite 8.2 (www.spotfire.com) was used for gene-profiling analysis, genes only present in exosomes and expression differences. For the network analysis to identify biological mechanisms, the program Ingenuity was used (www.ingenuity.com).
