All ultracentrifugations were performed at 120000 g AVG in Beckman Coulter Optima L-100 XP ultracentrifuge (stopping without break), with centrifugation durations based on a “50 nm cut-off size” adjustment to the centrifugation duration for each rotor as described in Livshits et al. 2015, with additional 5 min added to allow the rotor to come up to speed46 (link). Serum samples were defrosted and transferred to a 5 ml Beckman ultracentrifuge tube (Prod. No. 344057). The serum was then diluted to 4.6 ml with particle-free PBS and 400 µl of 40% iodixanol-PBS was pipetted into the bottom of the tube for Cushion ultracentrifugation (CUC) while the serum was diluted to 5 ml with particle-free PBS for ultracentrifugation (UC). The tubes were centrifuged at 120000 g (RCF avg, 35600 rpm) for 2 hours at 4 °C, using Beckman Coulter SW55ti rotor. For UC, the supernatant was removed and the EV pellet was resuspended in 50–100 µl residual PBS. For CUC the EV sample was resuspended in 5 ml particle-free PBS and centrifuged at 120000 g (RCF avg, 35600 rpm) for 2 hours at 4 °C, using Beckman Coulter SW55ti rotor. The supernatant was removed and the EV pellet was resuspended in 50–100 µl residual PBS.
Sw55ti rotor
Manufactured by Beckman Coulter
Sourced in United States, Germany, Canada
The SW55Ti rotor is a high-performance ultracentrifuge rotor designed for Beckman Coulter's line of ultracentrifuges. It is a swinging-bucket rotor capable of achieving high-speed centrifugation for a variety of research applications.
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Lab products found in correlation
Sw32ti rotor, by Beckman Coulter (10 mentions)
Optiprep, by Merck Group (8 mentions)
Piston gradient fractionator, by BioComp Instruments (7 mentions)
Tla 55 rotor, by Beckman Coulter (5 mentions)
Optiprep, by Beckman Coulter (4 mentions)
Optima l 100 xp, by Beckman Coulter (4 mentions)
293aav cells, by Cell Biolabs (4 mentions)
Sw28 rotor, by Beckman Coulter (4 mentions)
Ultra clear tube, by Beckman Coulter (4 mentions)
Exosome depleted fbs, by System Biosciences (4 mentions)
Sw41ti rotor, by Beckman Coulter (3 mentions)
5 ml centrifugation tube, by Beckman Coulter (3 mentions)
Gradient master, by BioComp Instruments (3 mentions)
Sw60ti rotor, by Beckman Coulter (3 mentions)
Ultracentrifuge, by Beckman Coulter (2 mentions)
Bio dot microfiltration apparatus, by Bio-Rad (2 mentions)
Histodenz, by Merck Group (2 mentions)
Gradient station, by BioComp Instruments (2 mentions)
Optima l 90k, by Beckman Coulter (2 mentions)
Ls 6500 multi purpose scintillation counter, by Beckman Coulter (2 mentions)
188 protocols using sw55ti rotor
1
Ultracentrifugation of Serum Extracellular Vesicles
2
Isolation and Purification of Tumor-Derived Extracellular Vesicles
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Following excision, BCa tissue samples were immediately immersed in 4 mL Dulbecco modified Eagle medium (Wako Pure Chemical Co.) without fetal bovine serum and stored at 4°C for 2 h. The tissue‐immersed medium was then centrifuged at 2000 g for 30 min at 4°C, and the collected supernatant was centrifuged at 17 000 g for 30 min at 4°C to remove cell debris and large EVs. The supernatant was filtered through a 0.22‐μm filter and transferred to a 5‐mL Ultra‐Clear Tube (Beckman Coulter), followed using ultracentrifugation at 100 000 g for 90 min at 4°C using an SW 55Ti rotor (ravg = 84.6 mm and adjusted k‐factor = 139.1; Beckman Coulter). The pellet was then washed with PBS and ultracentrifuged at 100 000 g for 90 min at 4°C using an SW 55Ti rotor (ravg = 84.6 mm and adjusted k‐factor = 139.1; Beckman Coulter), and the final pellet was resuspended in 100 μL PBS and frozen at −80°C. The procedure for Te‐EV secretion and isolation is shown in Figure 1B . The protein concentration of Te‐EVs was also measured using a Micro BCA protein assay kit (Thermo Fisher Scientific).
3
Production and Purification of Norovirus VLPs
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The GI.1 West Chester (2001, AY502016), GII.4 Sydney (X459908), GII.17 Kawasaki308 (2015, LC037415) VLPs were produced in Spodoptera frugiperda (Sf9) cells using baculovirus expression system as described previously (Koromyslova et al., 2017 (link)). In short, the recombinant VP1 bacmids were transfected into the Sf9 cells using Effectene (Qiagen). After 5 days of incubations at 27°C the cells were harvested, centrifuged, and the supernatant containing baculovirus was used to infect high five (H5) insect cells. The H5 cells were incubated at 27°C for 6 days. At 6 days post infection, the supernatant containing secreted VLPs was collected. The VLPs were concentrated by ultra-centrifugation at 35000 rpm (SW55 Ti rotor, Beckman Coulter) for 2 h at 4°C and purified by the CsCl equilibrium gradient ultracentrifugation at 35000 rpm (SW55 Ti rotor, Beckman Coulter) for 18 h at 4°C. The morphology of the VLPs was examined by electron microscopy.
4
Plasma Membrane Fractionation Protocol
5
Preparation of Outer Membrane Proteins
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The outer membrane protein (OMP) preparation was made essentially according to the method of Wang et al. (44 (link)) with some modifications. Briefly, cells were pelleted from 25-ml overnight cultures of all the strains (wild type and ΔvanT, ΔvanO, ΔompK, ΔvanT ΔompK, and ΔvanO ΔompK mutants), resuspended in 4.5 ml of water, and sonicated on ice (amplitude of 100, 3 min). To solubilize the cytoplasmic membranes, Sarkosyl (N-lauroylsarcosine) was added to a final concentration of 2% and incubated at room temperature for 30 min. To pellet the outer membranes, the mixture was ultracentrifuged (400,000 rpm for 1 h at 4°C; SW55 Ti rotor; Beckman). The pellet was washed with ice-cold water and ultracentrifuged again (400,000 rpm for 30 min at 4°C; SW55 Ti rotor; Beckman). The pellet was resuspended in 100 µl 100 mM Tris-HCl (pH 8) and 2% SDS buffer, and the proteins were separated on a 10% SDS-polyacrylamide gel and stained with Coomassie blue.
6
Purification of Virus and Extracellular Complexes
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Virus-infected HeLa cells or RNA-transfected L cells were freeze-thawed and clarified by differential centrifugation. The supernatant was collected, and virus was pelleted though a 30% (wt/vol) sucrose cushion at 300,000 × g (using a Beckman SW55 Ti rotor) for 3.5 h at 4°C. The virus pellet was resuspended in phosphate-buffered saline (PBS) and clarified by differential centrifugation. The supernatant was purified through a 15 to 45% (wt/vol) sucrose density gradient by ultracentrifugation at 300,000 × g (using a Beckman SW55 Ti rotor) for 50 min at 4°C (28 (link)). The peak fractions corresponding to virions and ECs were collected. The respective peak fractions were pooled and diluted, and the particles were pelleted by ultracentrifugation (using a Beckman SW55 Ti rotor) at 300,000 × g for 2 h at 4°C. The pellets were resuspended in 300 μl PBS and reclarified by centrifugation. The supernatants were repurified through a second 15 to 45% sucrose gradient at 300,000 × g for 50 min at 4°C, as described above.
7
Purification of G5 HEV Capsid Protein
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The culture supernatant of G5 HEV‐infected PLC/PRF/5 cells was collected at day 50 post inoculation (p.i.), and the cell debris were removed by centrifugation at 10,000g for 60 minutes, then concentrated by ultracentrifugation at 100,000g for 3 hours in a Beckman SW 55 Ti rotor. The resulting pellet was suspended in PBS at 4°C overnight, loaded onto a 10%‐50% (wt/vol) sucrose gradient, and centrifuged at 35,000g for 2 hours at 4°C in a Beckman SW 55 Ti rotor. The gradient was fractionated into 250 μL aliquots, and the G5 HEV capsid protein and HEV RNA in each fraction were detected by WB and RT‐qPCR, respectively.
8
SV40 Purification via OptiPrep Gradient Centrifugation
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SV40 was purified using the OptiPrep (60% stock solution of iodixanol in water; Sigma) gradient centrifugation method described previously in [39 (link)]. Briefly, viral genome transfected CV-1 cells were lysed in a buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl and 0.5% Brij 58 for 30 min on ice, and the supernatant was collected after centrifugation at 20,000x g for 10 min. The supernatant was placed on top of a discontinuous OptiPrep gradient of 20% and 40%, and centrifuged at 49,500 rpm for 2 h at 4°C in an SW55Ti rotor (Beckman Coulter, Indianapolis, IN). A white interface formed between 20% and 40% OptiPrep was collected, and aliquots were stored at -80°C for future use. Purified BKV and antibody against BKV large TAg (pAB416) were generous gifts from Dr. Michael Imperiale (University of Michigan).
9
Fractionation of Polysomes by Density Gradient
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For the fractionation of total polysomes, ribosomes (P170) were isolated as described above. Ribosome suspension was loaded on a 15–60% (w/v) sucrose density gradient (40 mM Tris–HCl pH 8.4, 20 mM KCl, 10 mM MgCl2 and 5 μg ml−1 cycloheximide) and centrifuged at 275 000 × g for 1.5 h at 4°C in an SW55 Ti rotor (Beckman Coulter). The UV absorbance profile at 254 nm was recorded using an ISCO 520 gradient system (ISCO, Lincoln, NE, USA). For immunoblot analysis, fractions were analyzed as described above.
10
Membrane-cytoplasm fractionation of HEK 293T cells
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HEK 293T Cells expressing C‐terminal GFP tagged FAM69C were grown to confluency in a 150 mm dish and treated with or without 0.5 mM sodium arsenite (AS) for 1 h. Membrane and cytoplasm were isolated with Applygene P1201. For density gradient centrifugation, cells were lysed in CER buffer (Applygene P1201) for 15 s. The lysate was then centrifuged for 5 min at 800 g. 1 ml Supernatant was layered on a linear sucrose gradient (15–45%, 400 μl/layer, 11 layer), centrifuged at 100,040 g in a Beckman sw55Ti rotor at 4°C for 40 min. Fractions 1–10 (500 μl each) were harvested from the top to the bottom. 15 μl from each fraction was analyzed by western blot. Fraction 1 represents the lowest density fraction, and fraction 10 represents the highest density.
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