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MB Sample ID: SA092199
| Local Sample ID: | 17Dec11_27exo |
| Subject ID: | SU001337 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
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Sample Preparation:
| Sampleprep ID: | SP001345 |
| Sampleprep Summary: | Exosomes were isolated from plasma by differential ultracentrifugation adapted from Refs. [47,48]. 0.7 mL cleared plasma (see above) were placed in 5 41 mm polyallomer ultraclear ultracentrifuge tubes on ice, and centrifuged for 1 h at 70,000 g at 4 C in a SWTi55 swing out rotor (Beckman). The supernatant was recentrifuged at 100,000 g for 1 h at 4 C, and the pellet was drained and resuspended in 0.7 mL cold PBS, and recentrifuged at 100,000 g for 1 h at 4 C. The washed exosomal pellets were resuspended in 100 mL nanopure water, vortexed for 30 s and transferred to a fresh microcentrifuge tube. The ultracentrifuge tube was washed with another 100 mL of nanopure water, vortexed for 30 s and the wash was transferred into same microcentrifuge tube, using the same pipet tip. The combined exosome suspensions were then lyophilized except for a small portion that was used for characterization by particle size distribution analysis (see below). These nanoparticles are operationally defined as exosomes. The lyophilized EXO preparations were extracted for lipidic metabolites using a solvent partitioning method with CH3CN:H2O:CHCl3 (2:1.5:1, v/v) as described previously [49]. The resulting lipid extracts were vacuum-dried in a vacuum centrifuge (Eppendorf), redissolved in 200 mL CHCl3:CH3OH (2:1) with 1 mM butylated hydroxytoluene, which was further diluted 1:20 in isopropanol/CH3OH/CHCl3 (4:2:1) with 20 mM ammonium formate for UHR-FTMS analysis. |
| Sampleprep Protocol Comments: | A small fraction (<1%) of each exosome preparation was characterized by size distribution analysis using a Nanosight 300 (Malvern Instruments), which provided the distribution of the Stokes' radius (mean 60e66 nm) and the number density of the particles. A typical analysis is shown in Fig. S1. The method eliminates very small particles, and provides a strongly peaked, narrow distribution at the expected size for exosomes (40e100nm, observed mode of 60e65 nm for the main peaks in Figs. S1A and B). |
