Beckman optima l 100xp

For exosome isolation, A375, B16 and B16F10 cells at 80% confluence were washed thrice with phosphate buffer solution (PBS) and then cultivated with growth medium containing 10% extracellular vesicles (EVs)-depleted FBS (prepared by overnight ultracentrifugation of medium-diluted FBS at 100,000 g at 4 °C). After 48 h, the conditioned medium (CM) was collected and pre-cleared by centrifugation at 800 g for 15 min and then at 10,000 g for 30 min. Exosomes were isolated by ultracentrifugation at 110,000 g for 70 min and washed in PBS by using the same ultracentrifugation conditions. Ultracentrifugation experiments were conducted with Beckman Optima L-100XP (Beckman Coulter, USA). Exosomes were observed by transmission electron microscopy HT7700 (HITACHI, Japan). The hydrodynamic diameter of exosomes was measured by using Nano-ZS ZEN 3600 (Malvern Instruments, UK).

S-CREM1 phage suspensions were inoculated into 2 L of exponentially growing cultures of Synechococcus sp. CB0101 at an MOI of 0.1. After host cell lysis, RNase A and DNase I were added to the lysates both at a final concentration of 2 µg mL⁻¹, and they were treated at room temperature for 1 h. Afterward, the NaCl concentration of phage lysates was adjusted to 1 M, and the lysates were ice-bathed for 0.5 h. To remove the remaining cells and debris, the phage lysates were centrifuged at 12,000× g at 4 °C for 20 min and further filtered through 0.22 µm filters (Millipore, Bedford, MA, USA). The filtrates were treated with PEG8000 (w/v 10%) and kept at 4 °C for 24 h [22 (link),33 (link)]. The PEG-treated phage suspensions were centrifuged at 12,000× g at 4 °C for 1 h to precipitate phage particles and then resuspended with 6 mL of TM buffer. Concentrated phage particles were then purified by CsCl density gradient ultracentrifugation (gradient density 1.45, 1.5, 1.55, and 1.6 g mL⁻¹, 200,000× g at 4 °C, 6 h) in a SW 41Ti rotor (Beckman Optima L-100XP, Beckman Coulter, CA, USA) [8 (link),34 (link)]. The visible phage band was extracted and then desalted using a 30 kDa centrifugal ultrafiltration unit. The purified high-titer phages were stored at 4 °C.

The EVs were isolated from seminal plasma by serial ultracentrifugation following a modification of the procedure described by Thery et al.^{46 (link)}. First, the ejaculates were centrifuged twice (1,500 × g for 10 min at room temperature [RT]; Rotofix 32 A; Hettich Centrifuge UK, Newport Pagnell, Buckinghamshire, England, UK) immediately after collection to obtain the bulk seminal plasma. Aliquots (5 mL) of seminal plasma were stored in cryotubes and sent in insulated containers with dry ice to the Andrology Laboratory of the Veterinary Teaching Hospital of the University of Murcia (Spain). At the laboratory, the seminal plasma samples were stored at -80 °C (Ultra Low Freezer; Haier Inc., Qingdao, China) for two weeks. After this storage period, the seminal plasma samples were thawed at RT inside a dark chamber and centrifuged at 10,000 × g for 30 min at RT to remove any possible remaining sperm- or other cell debris. The supernatants (2.5 mL) were centrifuged at 100,000 × g for 70 min at 4 °C using a swinging-Bucket rotor type SW41 Ti on a Beckman Optima L-100XP ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellets were then washed with 0.1 μm filtered-PBS and centrifuged at 100,000 × g for 70 min at 4 °C. The resulting pellets were resuspended in 0.1 μm filtered-PBS (50 μL) and stored at -80 °C in 5-μL aliquots (EVs-preparations) until analysis.

First, FBS was centrifuged at 11,000 g for 16 h to eliminate the exosomes; this supernatant was used as exosome-depleted FBS. Cells were maintained in complete medium until they reached 70-80% confluence. Then, the cells were transferred to medium with 10% exosome-free FBS. After 48 h, the supernatants were collected and centrifuged at 2,000 g for 10 min to remove the dead cells and debris and at 10,000 g for 30 min to remove the large extracellular vesicles (EVs). Next, the supernatants were concentrated with a 30KD ultrafiltration device (Millipore, Germany) and filtered through 0.22-µm filters (Millipore, Germany) to eliminate vesicles larger than 200 nm. The resulting supernatants were centrifuged at 110,000 g for 70 min in a Beckman Optima L-100 XP ultracentrifuge (Beckman Coulter, Germany). The exosome precipitates were washed with PBS and centrifuged for another 70 min. The concentrations of the exosomes were determined using a BCA protein assay kit (Thermo, USA). Finally, the exosomes were resuspended in PBS and stored at -80°C. The exosomes isolated from PANC-1 and MIA PaCa-2 cells were designated as Exo-Pan and Exo-Mia.

In total, 30 rats were used to isolate serum exosomes for the whole study. About 7ml whole blood could be collected from one sham rat via the abdominal aorta into a vacutainer. The whole blood was allowed to clot at room temperature for 30 min. The serum (1–2 ml) was obtained by centrifugation at 2,500 g for 10 min. The serum was ultracentrifuged at 100,000 g for 60 min (Beckman Optima L-100XP, Beckman, United States) and the pellets were resuspended in phosphate-buffered saline (PBS) and then ultracentrifuged at 150,000 g for another 90 min. The pellets were resuspended in 1 ml PBS and passed through the 0.22 μm filter (Millipore, SLGPR33RB) to obtain sterile exosomes. The protein concentration of exosomes was determined with bicinchoninic acid (BCA) commercial kit (Vazyme, China) as manufacturer’s instruction. The protein concentration of 1 ml exosomes is 0.1–0.2 mg/μl. The exosomes were aliquoted and stored at –80°C until use.
The serum exosomes (labeled as Con-exo) were observed using transmission electron microscopy (TEM) to identify the morphology. The size and concentration distribution profile of serum exosomes were analyzed using a Multiple-Laser ZetaView^® f-NTA Nanoparticle Tracking Analyzers (Particle Metrix, Germany). The exosome surface markers were confirmed by Western blotting using antibodies against CD9 and TSG101.

The acquired bitter melon was washed with deionized water and juiced using a juicer. The juice was centrifuged at 3000× g for 30 min, 10,000× g for 40 min at 4 °C (Centrifuge 5804 R, Eppendorf, Hamburg, Germany). After two centrifugations, the color of the supernatant changed from green to transparent. A 0.22 μm filter was used to remove the remaining impurities from the supernatant. Then, 1 mL Opti-prep was added to each tube’s bottom. The supernatant was centrifuged at 100,000× g for 90 min at 4 °C (Beckman Optima L-100XP, Beckman, Brea, CA, USA), and a 1 mL sample was collected from the tube’s bottom. All collected liquid was combined and ultracentrifuged under the same conditions. The pellets were suspended in sterile phosphate-buffered saline (PBS), and the resulting BMVE was frozen at −80 °C for future use.

CRC cells were cultured in RPMI‐1640 medium containing with 10% exosome‐free FBS. FBS was depleted of exosomes by ultracentrifugation using Beckman Optima L‐100XP (Beckman Coulter, USA) at 110 000 × g at 4°C overnight. Cell culture medium was collected after 72 h and centrifuged at 2000 g for 20 min and 2 5000 g for 40 min at 4°C. The supernatant was filtered with a .22 μm filter and centrifuged at 110 000 × g for 90 min at 4°C. Harvested exosomes were quantified by the BCA Protein Assay kit (Millipore, Billerica, MA, USA). Exosomes were observed and identified on transmission electron microscopy HT7700 (HITACHI, Japan). Exosomes were labelled with PKH67 (Sigma, St. Louis, MO, USA) as previously described.⁵⁰ The exosomes were added with 10 ml PBS, collected by ultracentrifugation at 110 000 × g for 90 min, then resuspended in PBS and then incubated with HUVEC for 240 min and photographed by a fluorescence microscope. For cell processing, the number of recipient cells was controlled to 2 × 10⁵ and exosomes and recipient cells were incubated for 72 h. CM of EMT‐HCT116/SW620 cells treated with RNase (2 mg/ml) alone or with Triton X‐100 (.1%)