EVs were isolated and characterized according to the 2018 consensus statement on minimal information for studies of extracellular vesicles (MISEV2018) (6 (link)). Cells were cultured for 48 h in DMEM supplemented with 10% exosome depleted FCS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% penicillin-streptomycin. Supernatants (30 ml) were collected and centrifuged for 10 min at 300 g to remove cells and cell debris, followed by 30 min at 10000 g to remove larger vesicles. Afterwards the supernatants were filtered through a 0.2 µm filter and centrifuged at 100000 g for 1.5 h. Pelleted EVs were washed with PBS and centrifuged for another 1.5 h at 100000 g. Centrifugation was performed using a Sorvall WX+ Ultra Centrifuge, with SureSpin 632 rotor (k-factor 194) (Thermo Fisher Scientific, Waltham, Massachusetts, USA). EVs were resuspended in PBS. EV analysis was performed by nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern Panalytical, Kassel, Germany). Therefore, EVs were isolated or samples were diluted (conditioned media (1:100) or patient serum (1:1000)) without isolation and analysed from three independent biological samples. Measurements were performed at a controlled temperature of 22°C. For each sample, three measurements of 30 s were performed. EV concentration and size was calculated by the NanoSight software.
Sorvall wx ultracentrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sorvall WX+ Ultracentrifuge is a high-speed centrifuge designed for laboratory applications. It is capable of achieving centrifugal forces up to 1,000,000 x g, allowing for the separation and purification of various biomolecules, cells, and sub-cellular components. The Sorvall WX+ features a compact and ergonomic design, as well as advanced temperature control and safety features to ensure reliable and consistent performance.
Automatically generated - may contain errors
Lab products found in correlation
Exosome depleted fcs, by Thermo Fisher Scientific (1 mentions)
Surespin 632 rotor, by Thermo Fisher Scientific (1 mentions)
Nanosight ns300, by Malvern Panalytical (1 mentions)
Dnase 1, by Zymo Research (1 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
Celltracker red cmtpx, by Thermo Fisher Scientific (1 mentions)
Netwell inserts, by Corning (1 mentions)
Nigericin, by Merck Group (1 mentions)
Nigericin, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Pierce microplate bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Surespin 630 swinging bucket rotor, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Staining buffer, by BioLegend (1 mentions)
Pkh67, by Merck Group (1 mentions)
0.22 m filter, by Merck Group (1 mentions)
0.22 mm filter, by Merck Group (1 mentions)
T 865 fixed angle rotor, by Thermo Fisher Scientific (1 mentions)
Centrifuge 5804 r, by Eppendorf (1 mentions)
12 protocols using sorvall wx ultracentrifuge
1
Isolation and Characterization of Extracellular Vesicles
2
HEV Detection in Pig Fecal Samples
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Eighteen stool samples (10–15 gr) were collected in 2020 in sterile bags from a small-sized farm situated in Salto City, where the highest seroprevalence of HEV had been reported in a previous study [16 (link)]. Samples were kept on dry ice and sent to the laboratory where they were processed.
Pig fecal samples were resuspended at 10% w/v in sterile phosphate buffer saline (PBS) and vigorously vortexed. After centrifugation for 30 min, 8000× g at 4 °C, the supernatant was filtered to 0.22 μM. Total RNA was extracted with a Quick-RNA™ Miniprep Kit and treated with DNase I (Zymo Research Corp., Tustin, CA, USA) according to the manufacturer’s instructions.
For the detection of HEV, RNA was subjected to RT-nPCR targeting a 330-bp region within ORF2 as previously reported [17 (link)]. PCR products were gel-visualized under UV light and amplicons of the expected size were sequenced in both directions by Macrogen Inc. (Seoul, Republic of Korea).
The HEV-positive samples were filtered (0.45 μM) and viral particles were concentrated in a 30% sucrose gradient and ultra-centrifuged for 2:30 h at 100,000× g, 4 °C in a Sorvall™ WX+ Ultracentrifuge (Thermo Scientific, Waltham, MA, USA). The pellet was resuspended in 200 µL of PBS 1× and RNA was then extracted as mentioned above.
Pig fecal samples were resuspended at 10% w/v in sterile phosphate buffer saline (PBS) and vigorously vortexed. After centrifugation for 30 min, 8000× g at 4 °C, the supernatant was filtered to 0.22 μM. Total RNA was extracted with a Quick-RNA™ Miniprep Kit and treated with DNase I (Zymo Research Corp., Tustin, CA, USA) according to the manufacturer’s instructions.
For the detection of HEV, RNA was subjected to RT-nPCR targeting a 330-bp region within ORF2 as previously reported [17 (link)]. PCR products were gel-visualized under UV light and amplicons of the expected size were sequenced in both directions by Macrogen Inc. (Seoul, Republic of Korea).
The HEV-positive samples were filtered (0.45 μM) and viral particles were concentrated in a 30% sucrose gradient and ultra-centrifuged for 2:30 h at 100,000× g, 4 °C in a Sorvall™ WX+ Ultracentrifuge (Thermo Scientific, Waltham, MA, USA). The pellet was resuspended in 200 µL of PBS 1× and RNA was then extracted as mentioned above.
3
Isolation of Placental Extracellular Vesicles
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Placental large and small EVs were collected from human term placentae, using a well-established explant culture model [48 (link)]. Briefly, the placental tissues were dissected into explants of 400 mg wet weight and washed thoroughly to remove the contaminating blood. Then, the explants were cultured in 12-well culture plates with Netwell inserts (400 µm, Corning, Glendale, AZ, USA) in Advanced DMEM/F12 medium (In Vitro Technologies, Auckland, NZ), complemented with 2% FBS and 1% P/S overnight. The cultured medium was collected and centrifuged sequentially at 4 °C as follows: 2000× g for five minutes to remove the macro-vesicles/syncytial nuclear aggregates (SNAs) and cellular debris, 20,000× g for 60 min to collect large EVs, and 100,000× g for 60 min to collect small EVs (Sorvall wX+ ultracentrifuge (Thermo Fisher, San Jose, CA, USA)). The EV pellets were resuspended in PBS and were stored at 4 °C for future use.
In some experiments, the human placental explants were cultured in the presence of CellTrackerTM Red CMTPX (1 µg/mL, Thermo Fisher, Eugene, OR, USA) to generate fluorescently labelled placental large and small EVs.
In some experiments, the human placental explants were cultured in the presence of CellTrackerTM Red CMTPX (1 µg/mL, Thermo Fisher, Eugene, OR, USA) to generate fluorescently labelled placental large and small EVs.
4
Cell Fractionation and Membrane Protein Extraction
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RAW cells were lysed in 200 μl ice cold homogenization buffer (3 mM imidazole, 250 mM sucrose, 0.5 mM EDTA, pH 7.4 with protease inhibitor cocktail). Cells were homogenized by passing 10x though a 25-gauge needle, then lysates were sequentially centrifuged at 3000 xg for 10 min at 4°C and 7,000 xg for 10 min at 4°C to clear supernantants. Supernatants were then further centrifuged at 100,000 xg using SORVALL wX+ULTRA-centrifuge (Thermo Scientific) for 30 min at 4°C to separate cytosol and membranes. Next, the pellets were resuspended in 0.5% digitonin in solubilization buffer (50 mM NaCl, 50 mM imidazole, 2.5 mM 6-aminohexanoic acid, 2 mM EDTA, pH ~7) to obtain membrane-bound materials.
5
Isolation and Characterization of Extracellular Vesicles
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After 24-hour culture of 1 × 106 cells/mL HERV-K dUTPase or GFP overexpressing THP-1 monocytes in 30 mL RPMI1640 media with exosome-depleted 10% FBS (Gibco), THP-1 monocytes were primed with 1 μg/mL LPS (Sigma-Aldrich) for 3 hours and treated with 10 μM nigericin (Sigma-Aldrich) for an additional 1 hour to stimulate the release of EVs. After that, the culture media was collected and precleared by centrifugation at 300g for 10 minutes, then at 2000g for 10 minutes to eliminate dead cells and cellular debris. The supernatant was then ultra-centrifuged at 100,000g for 70 minutes at 4°C (Sorvall WX+ Ultracentrifuge, Thermo Fisher Scientific), followed by washing of the EV pellet with PBS at 100,000g for 70 minutes at 4°C to remove LPS and nigericin. To characterize the collected EVs, pellets were resuspended in saline, and the number and size of the particles measured by NanoSight (Malvern Panalytical). The concentration of the particles (particles/mL) was used to determine the volume of the injected suspension to achieve the desired dosing (particles/g BW) in the mice. For western blot analysis, we normalized for EV quantity based on the protein content of the EVs, determined using Pierce microplate BCA protein assay kit (Thermo Fisher Scientific).
6
Generating Retrovirus in HEK293T Cells
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RV-GFP was done as described before [116 (link)]. HEK293T cells were co-transfected with pCAG-GFP, pCMV-GP, and pCMV-VSV-G (3:2:1) plasmids by calcium-phosphate precipitation. The media containing retrovirus was collected 48 h after transfection. Cell debris was removed from the supernatant by centrifugation at 3200×g for 10 min and filtration through a 0.22 μm filter. The retrovirus was concentrated by ultra-centrifugation at 160,000×g for 2 h (Sorvall WX Ultracentrifuge and SureSpin 630 swinging bucket rotor; Thermo Fisher Scientific, Waltham, MA, USA). The retroviral pellet was resuspended in 200 μl phosphate buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA), aliquoted and stored at −80 °C. The titer was at 105 colony forming units.
7
Fluorescent Labeling of Extracellular Vesicles
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We labeled 5 μg of EVs with the green lipophilic fluorescent dye PKH-67 (Sigma-Aldrich, USA) as previously described with some modifications (Liu et al., 2018 (link)). Briefly, we transferred 5 μg of EVs to 12 ml of 1640 medium in a conical-bottom polypropylene tube. EVs were centrifuged at 110,000×g for 90 min at 4°C in a Sorvall WX+ Ultracentrifuge (Thermo Scientific) with rotor T-1270 and to precipitate EVs. Subsequently, the supernatant was carefully aspirated. We added 1 ml of Diluent C to the EV pellet to prepare a 2× EV suspension solution. We mixed 1 ml of Diluent C with 2 μl of PKH67 to prepare 2× Dye Solution. The both were rapidly mixed, and the mixture was incubated for 4 min at room temperature. The labeling reaction was stopped by adding an equal volume of 1% BSA to the labeling solution. Excess dye was removed by ultracentrifugation at 110,000×g for 90 min with RPMI 1640 medium. To verify the internalization of EVs by spleen immune cells, we labeled 5 μg of EVs from different groups (i.e., plasma, PCS, and HCF) with PKH-67 and added them to 5 × 105 spleen mononuclear cells, which were incubated for 4 h at 37°C and 5% CO2. Afterward, we collected and washed the cells with a staining buffer (Biolegend, USA).
The strategy gates for immune cell analysis are shown inSupplementary Figure 2
The strategy gates for immune cell analysis are shown in
8
Sucrose Gradient Fractionation of Digested Samples
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The digested samples were then loaded on top of previously prepared sucrose gradients (gradient 5% to 50% sucrose in 5% steps and 1 mL volume per step) and separated by ultracentrifugation (Sorvall WX+ Ultracentrifuge from Thermo Scientific with a SW41 Ti-Rotor) overnight (18 h, 110,000–115,000× g, 4 °C). The gradient was fractionated in 395 µL aliquots (25 fractions per gradient). The samples were snap-frozen in liquid nitrogen and stored at −80 °C for later use for Western blot validation or mass spectrometry analysis. For a detailed protocol, refer to [30 (link)].
9
Serum Exosome Isolation Protocol
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Blood samples from a vein in the patient’s forearm were collected and placed into blood collection tubes, which were centrifuged at 2400 × g for 10 minutes to isolate the serum. Serum was aliquoted and stored at –80°C. Serum exosomes were isolated by sequential ultracentrifugation in accordance with a published procedure (Momen-Heravi et al., 2013; Momen-Heravi, 2017). Briefly, cell contamination was removed from blood by centrifugation at 300 × g for 10 minutes, and the remaining sample was centrifuged at 2000 × g for 10 minutes to eliminate apoptotic bodies and large cellular debris. The remaining sample was then centrifuged at 10,000 × g for 30 minutes to remove large micro-vesicles. After centrifugation, the supernatant was filtered using a 0.22-μm microporous filter head (Millipore, Bedford, MA, USA) in 10-mL ultracentrifugation tubes (Thermo Fisher Scientific, Waltham, MA, USA). Exosomes were then collected by ultracentrifugation at 100,000 × g for 70 minutes and were subsequently washed in phosphate-buffered saline (PBS) and pelleted again by ultracentrifugation at 100,000 × g in a T-890 rotor (Sorvall WX+ ultracentrifuge, Thermo Fisher Scientific). All centrifugation steps were performed at 4°C. The exosome pellet was resuspended in 30 µL of PBS for immediate use or stored at –80°C until further experimentation was performed.
10
Exosome Isolation and DNase Inactivation
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Three-day-conditioned media from HEK cells cultured in exosome-depleted medium were harvested and centrifuged at 300 g for 25 min. Supernatants were filtered with 0.22 µm filters (Merck Millipore, Burlington, MA, USA) and centrifuged in a SorvallTM WX Ultracentrifuge (ThermoFisher Scientific, Waltham, MA, USA, WX Ultra 100 #75,000,100) at 150,000× g for 2 h at 4 °C with a SureSpinTM 630 swinging bucket rotor (ThermoFisher Scientific) to pellet EV. After supernatant was carefully removed, the EV-containing pellet was resuspended in PBS and stored at −80 °C until use.
The isolated sEVs were aliquoted and submitted to three different protocols. Thirty micrograms of sEVs were treated with 1 μL of DNase I, 1 μL of reaction buffer and then incubated and heated as described above for the DNase I treatment procedure. As a control, thirty micrograms of sEVs were treated in same way, excepted the supplementation of DNase I and heated at 65 °C for 10 min to consider the thermal effect on isolated sEVs (thermal treatment), while the last aliquot of exosomes remained untreated and used as control.
The isolated sEVs were aliquoted and submitted to three different protocols. Thirty micrograms of sEVs were treated with 1 μL of DNase I, 1 μL of reaction buffer and then incubated and heated as described above for the DNase I treatment procedure. As a control, thirty micrograms of sEVs were treated in same way, excepted the supplementation of DNase I and heated at 65 °C for 10 min to consider the thermal effect on isolated sEVs (thermal treatment), while the last aliquot of exosomes remained untreated and used as control.
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