To examine the uptake of SP-EVs by COCs, SP-EV subsets were labelled with PKH67 Green Fluorescent Cell Linker kit (MIDI67), a green-fluorescent dye that labels membrane lipids and is widely used for visualisation of EVs uptake by cells73 . The EVs were stained following the protocol described by Almiñana et al.74 with slight modifications. Briefly, each EV-sample (S-EV and L-EV; 25 µL) was mixed with diluent C (1 µL). Then, PKH67 (1 µL) was diluted in diluent C (100 µL), and the resulting volume was added to the previously prepared sample. After 5 min of incubation at room temperature, EV-depleted foetal bovine serum (FBS; 250 µL) was added to the mixture and incubated for 1 min to stop the labelling. This EV-depleted FSB was prepared the previous day by ultracentrifugation of heat inactivated FBS (ref. 10,500–056; Gibco, Thermo Fisher Scientific) at 100,000 xg and 4 °C for 16 h (Optima L-100 XP Ultracentrifuge using rotor SW55; Beckman Coulter, CA, USA). Two washes were made by filling 1-mL ultracentrifugation tubes (50 Ultra-Clear™ tubes; Beckman Coulter) with filtered PBS and subsequent ultracentrifugation at 100,000 xg and 4 °C for 30 min. The SP-EV labelled pellet was resuspended in IVM medium and stored at 4 °C until IVM was performed the next day following the aforementioned protocol.
Ultra clear tube
Manufactured by Beckman Coulter
Sourced in United States
Ultra-Clear tubes are a line of laboratory tubes designed for sample collection, processing, and storage. They are made of transparent material, allowing for visual inspection of the contents. The tubes are available in a range of sizes and are suitable for use in a variety of laboratory applications.
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Lab products found in correlation
Optima l 100 xp ultracentrifuge, by Beckman Coulter (9 mentions)
0.22 μm filter, by Merck Group (8 mentions)
Sw28 rotor, by Beckman Coulter (5 mentions)
Optima xl 70, by Beckman Coulter (5 mentions)
Sw41ti rotor, by Beckman Coulter (5 mentions)
Sw55ti rotor, by Beckman Coulter (4 mentions)
Optima l 80 xp, by Beckman Coulter (3 mentions)
Optima l 100 xp, by Beckman Coulter (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Optima xe 100, by Beckman Coulter (3 mentions)
Sw41 t1, by Beckman Coulter (3 mentions)
Phosphate buffered saline (pbs), by AppliChem (3 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (3 mentions)
Nunc easydish dishes, by Thermo Fisher Scientific (2 mentions)
Falcon 50 ml conical centrifuge tubes, by Thermo Fisher Scientific (2 mentions)
Zetaview pmx 110, by Particle Metrix (2 mentions)
Sw28 swinging bucket rotor, by Beckman Coulter (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Optiprep, by Merck Group (2 mentions)
Optima xl 70 ultracentrifuge, by Beckman Coulter (2 mentions)
80 protocols using ultra clear tube
1
Labeling SP-EVs with PKH67 for COC Uptake
2
Extracellular Vesicle Isolation from Cell Culture
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HepG2, Hep3B, SNU387 cells were cultured in 18 Nunc EasYDish dishes (145 cm2, Thermo Fisher Scientific) for 72 h. Then the culture medium was switched to serum-free culture medium (Thermo Fisher Scientific) to starve the cells for 24–48 h. The serum-free culture medium incubated with cells was finally collected for EV isolation. After first centrifugation at 300 × g (4 °C) for 10 min to remove cells and cell debris, the supernatant was collected and transferred to new tubes and centrifuged at 2800 × g (4 °C) for 10 min to further eliminate the remaining cellular debris and large particles. The supernatant was carefully transferred to Ultra-Clear Tubes (38.5 mL, Beckman Coulter, Inc., USA), followed by ultracentrifugation using an Optima L-100 XP Ultracentrifuge (Beckman Coulter, Inc, USA) at 100,000 × g (4 °C) for 70 min. The EV pellets at the bottom of the tubes were carefully collected and resuspended in 200 µL fresh PBS. In control experiments, the EV pellets collected in 200 µL PBS were treated with RNase61 (link),62 (link) at 37 °C for 30 min first, then PBS was added to wash and recollect the EV pellets by ultracentrifugation at 100,000 × g (4 °C) for 70 min. For the artificial plasma samples, each 10 µL aliquot of EV pellets was spiked into 90 µL healthy donors’ plasma or cirrhotic patients’ plasma.
3
Purification and Characterization of Parvovirus Extracellular Vesicles
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Aliquots of supernatant 2 were layered onto 20% to 50% sucrose 10-step gradients made in Beckman Ultra-Clear tubes (5 by 41mm) and spun at 22,000 rpm in an SW 55Ti Beckman rotor for 45 min at 4°C. Fractions (1 drop each) were collected from the bottom of the centrifuge tube. Each fraction was diluted with PBS and examined by negative-staining EM to confirm the presence of PEVs. PEVs banded in the same location as mature virus, midway between the B-capsids and C-capsids seen in companion gradients (see Fig. S1 in the supplemental material). PEV-rich fractions were diluted with PBS and recentrifuged in 20% to 50% sucrose gradients as described above. Fractions were subjected to SDS-PAGE followed by Western blotting and probed (47 (link)) with pUL31-specific polyclonal antibody (Ul31-GST; a gift from Joel Baines, Louisiana State University [LSU] School of Veterinary Medicine) at a dilution of 1/2,000.
4
Cesium Chloride Gradient Purification
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A further purification of the isolated phage particles was conducted as preparation for the proteomics measurements. For this purpose, cesium chloride (CsCl) was dissolved in SM buffer creating solutions of different densities (1.3, 1.4, 1.5, 1.6 g/mL). These solutions were subsequently layered into a centrifuge tube with 2 mL per density (with lowest density on top). 2 mL of pre-purified phage solution (in SM buffer) were added on top of this gradient and centrifuged using open Ultra-Clear Tubes (Beckman Instruments Inc., Palo Alto, CA, USA) in a SW 28.1 swinging rotor (Beckman) with a Beckman Coulter Optima L-80XP ultracentrifuge (Beckman Coulter, Brea, CA, USA) at 150,000× g and 4 °C for 2 h.
During this centrifugation, a milky layer of phage particles appeared between the 1.3 g/mL and 1.4 g/mL layer. This milky layer was extracted, washed three times with SM buffer using Amicon Ultra centrifugal filters with 30 kDa cutoff (MerckMillipore, Burlington, MA, USA) and subsequently tested for their phage titer.
During this centrifugation, a milky layer of phage particles appeared between the 1.3 g/mL and 1.4 g/mL layer. This milky layer was extracted, washed three times with SM buffer using Amicon Ultra centrifugal filters with 30 kDa cutoff (MerckMillipore, Burlington, MA, USA) and subsequently tested for their phage titer.
5
Lentiviral Transduction of Mouse Models
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Lentiviruses were produced as described44 (link). Briefly, transient calcium phosphate cotransfection of HEK-293 cells was done with the pGIZP empty, pGIZP.shMkk6, pGIZP.shAmpk, pGIZP.shTak1, pGIZP.shTab1, or pGIZT.shMapk14 vectors from Thermo scientific together with pΔ8.9 and pVSV-G. The supernatants containing the LV particles were collected 48 and 72 h after removal of the calcium phosphate precipitate, and were centrifuged at 700 × g, 4 °C for 10 min, and concentrated (165×) by ultracentrifugation for 2 h at 121986 g at 4 °C (Ultraclear Tubes, SW28 rotor and Optima L-100 XP Ultracentrifuge; Beckman). Viruses were collected by adding cold sterile PBS and were titrated by quantitative PCR.
Mice were injected in VMH or tail vein with lentiviral particles suspended in PBS. Seven days after infection, mice were fed a HFD diet.
Mice were injected in VMH or tail vein with lentiviral particles suspended in PBS. Seven days after infection, mice were fed a HFD diet.
6
Purification and Quantification of HSV
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HSV virus stocks were obtained after purification on OptiPrep gradients (Axis Shield) similar to that described in Shah et al.26 (link) In brief, Vero cells were infected at low MOI (0.01). Both culture supernatants and cells were collected separately once infection had reached a 100% cytopathic effect (CPE). Supernatants underwent three centrifugation steps: step 1, 1,811 × g (15 min); step 2, transfer of supernatants to Nalgene tubes and centrifugation at 3,568 × g (5 min, JA14 rotor); and step 3, transfer of the final supernatants to Beckman Coulter Ultra-Clear tubes and centrifugation at 53,000 × g (1 hr, SW28 rotor). Viral pellets were resuspended in PBS containing 10% glycerol. Cells were scraped, lysed in 0.4 M NaCl, and subjected to multiple centrifugations as described above prior to suspension in PBS/10% glycerol. Virus pellets were purified on OptiPrep gradients as follows. Equal volumes of OptiPrep were added to NaCl-extracted or culture supernatant virus pellets, mixed, and transferred to Quick-Seal polyallomer tubes. Tubes were then filled with 25% OptiPrep and centrifuged for 3 hr at 354,000 × g in an NVT90 rotor. After centrifugation, virus fractions were collected, diluted in PBS, and centrifuged. Virus pellets from supernatants and cell lysates were combined, and the titer was determined on Vero cells as described previously.
7
Glycerol Gradient Ultracentrifugation
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One millilitre layers of glycerol (35–15%) were loaded in Ultraclear tubes (Beckman). Ultracentrifugation was performed in a Sw55ti rotor at 40 000 rpm for 8 h at 4°C. In all, 200 µl fractions were collected from the top of the gradient. An equal volume of each fraction was analysed by immunoblotting using the indicated antibodies.
8
Isolation and Characterization of Extracellular Vesicles from ES Cells
9
Exosome Isolation from Serum Samples
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The initial volume of serum for the experiments was 5 mL per group. The samples were diluted with an equal volume of Phosphate Buffered Saline (PBS) to decrease the viscosity. The diluted serum samples were centrifuged at 2000 g for 10 minutes, 10,000 g for 30 minutes at 4°C to remove dead cells and cell debris. The supernatant was transferred into Ultra-Clear tubes (Beckman Coulter, Indianapolis, IN), and centrifuged at 100,000 g using a Beckman Optima XL-70 ultracentrifuge for 70 minutes at 4°C. The supernatant was removed by pipette, and the supernatant was remained 2 mm above the pellet. Five cycles of ultracentrifugation were needed to purify exosomes and remove serum proteins.[12 (link)] After centrifugation, the exosomes appeared as beige in the bottom of the vessel. The exosomes dissolved in 200 μL SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0), and boiled for 5 minutes. Undissolved debris was removed by centrifugation at 14,000 rpm for 15 minutes. The supernatant were collected and quantified with a BCA Protein Assay Kit (Bio-Rad, CA, USA).
10
Isolation of hBMSC-derived Exosomes
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Exosomes, including Exo and MT-Exo derived from hBMSCs, were isolated from the supernatant via ultracentrifugation. Specifically, hBMSCs were pretreated with MT at a final concentration of 1 μmol/L in serum-free culture medium for 48 h. When the cell confluence reached 80%, the medium was harvested for centrifugation at 300g and 2000g to remove dead cells for 15 min and 20 min, respectively. Then, we filtered the harvested supernatant by a 0.22-μm filter (Micropore). Subsequently, Ultra-Clear™ tubes (Beckman Coulter, USA) were utilized for the filtration of supernatant at 100,000g for approximately 2 h twice. Finally, we used PBS for resuspending the required pellets before being kept at − 80 °C for further experiments.
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