Dulbecco s pbs

The supernatants were thawed and centrifuged again, and 130 μl of the supernatant was transferred to the micro cuvette (Starna Cell Inc., Atascadero, CA) for measurement. The fluorescence intensity in each sample was determined for Cy5 (λ_ex = 645 nm, λ_em = 662 nm) using an RF5301PC spectrofluorophotometer running Panorama 3 software (Shimadzu Scientific Instruments, Columbia, MD). The background fluorescence was adjusted from the fluorescence values of healthy control tissue. The fluorescence intensity values were then converted to dendrimer concentrations using calibration curves of PEGOL-60-Cy5 for appropriate slit widths. Plasma was diluted 10-fold in Dulbecco’s PBS (Corning Inc.), then passed through a 0.2-μm pore polyethersulfone (PES) filter, and measured as described above for organ samples. Tissue concentrations are presented as %ID/g tissue, which were calculated by dividing the converted PEGOL-60 mass from the calibration curves by the organ mass and initial dose and multiplying by 100 to convert to percentages.

Immediately after plasma preparation, exosomes were collected using an ExoQuick Plasma Exosome Isolation kit per manufacturer instructions (System Biosciences, EXOQ5TM). In brief, debris and nonexosomal particles were removed by a 15-minute incubation with thrombin at 37°C followed by centrifugation at 10,000 × g for 20 minutes. ExoQuick precipitation solution was added to each supernatant (1/3 v/v) and mixed by inversion. Samples were incubated overnight at 4°C and pelleted by centrifugation at 10,000 × g for 30 minutes at 4°C. The exosome-depleted supernatant was removed and the pellet was centrifuged at 10,000 × g for an additional 5 minutes at 4°C to remove remnant supernatant and ExoQuick reagent. Pellets were resuspended in sterile Dulbecco’s PBS (Corning) for size distribution and transmission electron microscopy (TEM) morphological analyses or sonicated in RIPA buffer with HALT protease inhibitor cocktail (Thermo Fisher) for protein analysis, then stored at −80°C.

Nanoparticle tracking analysis (NTA) was performed with a ZetaView nanoparticle analyzer (Particle Metrix GmbH; Germany). The 102-nm polystyrene beads were used to calibrate the apparatus before each session. We recorded 11 measurements for each case twice and the results were averaged in at least 1 dilution in Dulbecco’s PBS (Corning, Inc., NY, USA). ZetaView Software 8.04.02 (Particle Metrix GmbH) was used to analyze images, settings were set to default image evaluation, and we used the following camera acquisition parameters: sensitivity set to 85, shutter set to 70, and frame rate set to 30. Each sample was measured 3 times. Concentrations of the samples of extracellular vesicles were expressed as the number of EVs per 1 ml of diluents; in this case, PBS.

For immunoblots, cells were plated in 24-well dishes or 12-mm Trans­well filters (Corning) and grown for 6–10 d. Cells were washed twice in ice-cold Dulbecco’s PBS (Corning) and lysed in 0.10–0.25 ml of 4X SDS sample buffer (250 mM Tris [Sigma, 93362], pH 6.8, 40% glycerol [Sigma, G8773], 0.52 M β-mercaptoethanol [Calbiochem, 4203]) and 8% SDS [Sigma, L3771]). Lysates were sonicated briefly to shear genomic DNA, heated to 95°C for 3 min, and resolved by SDS–PAGE. Gels were transferred to nitrocellulose membranes (Bio-Rad, 1620115) and then blocked in a solution of PBS and 10% nonfat dry milk powder (NFDM; Carnation) for 1 h. Filters were incubated for 1 h in primary antibodies diluted in a solution of PBS (Corning, 10X, 46-013-CM), 5% NFDM, and 0.1% Tween-20 (Affimetrix, T1003)(PBS-T); washed four times for 5 min each in PBS-T; and incubated for 30 min with 1:2000 dilution of the appropriate species-specific secondary antibodies coupled to IRDyes (Rockland, Gilbertsville, MD). After four more washes in PBS-T, filters were imaged with the Odyssey infrared imaging system (Licor Biosciences, Lincoln, NE).