Sw41 swinging bucket rotor

Manufactured by Beckman Coulter

Sourced in Germany

The SW41 swinging bucket rotor is a laboratory equipment used for centrifugation. It is designed to separate and isolate biological samples, such as cells, organelles, and macromolecules, based on their sedimentation properties. The rotor features a swinging bucket design, which allows the samples to be evenly distributed and subjected to uniform force during the centrifugation process.

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Lab products found in correlation

Ti50.2 fixed angle rotor, by Beckman Coulter (2 mentions) Tube slicer, by Beckman Coulter (2 mentions) Ultraclear ultracentrifuge tubes, by Beckman Coulter (2 mentions) Nanosight ns500, by Malvern Panalytical (1 mentions) H 7650, by Hitachi (1 mentions) Amt ccd camera, by Advanced Microscopy Techniques (1 mentions) Ja 20 rotor, by Beckman Coulter (1 mentions) Jla 10.500 rotor, by Beckman Coulter (1 mentions) Ss 34fixed angle rotor, by Thermo Fisher Scientific (1 mentions)

13 protocols using sw41 swinging bucket rotor

Isolation and Characterization of Extracellular Vesicles

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EVs were isolated by ultracentrifugation of the secretome of irradiated and non-irradiated human PBMCs. To obtain EVs, secretomes were centrifuged at 20,000 g for 20 minutes and at 110,000 g for 120 minutes at 4 °C in an SW 41 swinging bucket rotor (Beckman Coulter). For lipid, protein, and RNA analysis, irradiated and non-irradiated PBMCs were cultured in pre-centrifuged (220,000 g for 24 hours at 4 °C) serum-free CellGenix to deplete EVs from the culture medium. The absolute number and size of EVs were assessed using a NanoSight, NS500 instrument (Malvern Instruments, Malvern, UK). The nanoparticles were visualized by laser light scattering and the motion of each nanoparticle tracked from frame to frame by NTA3.1 software. Recorded videos were analyzed by the NTA3.1 software. For comparability, isolated EVs were employed in the same concentrations as EVs present in MNC^aposec.

Wagner T., Traxler D., Simader E., Beer L., Narzt M.S., Gruber F., Madlener S., Laggner M., Erb M., Vorstandlechner V., Gugerell A., Radtke C., Gnecchi M., Peterbauer A., Gschwandtner M., Tschachler E., Keibl C., Slezak P., Ankersmit H.J, & Mildner M. (2018). Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions. Scientific Reports, 8, 18016.

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Sedimentation Analysis of Ty1 Gag Complexes

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Sedimentation of Gag complexes was analyzed as described (Tucker et al. 2015 (link)), with the following modifications. For strains BY4742 and HWA215, a 100-ml culture was grown as described for FISH/IF microscopy. Equal amounts of total protein (7–8 mg in 300–450 μl) were applied to a 7–47% continuous sucrose gradient and centrifuged at 25,000 rpm in a SW 41 swinging bucket rotor (Beckman Coulter, Brea, CA) for 3 hr at 4°. Nineteen 0.6-ml fractions were collected, and an equal volume of each fraction was subjected to western blot analysis to detect Ty1 Gag as described above.

Ahn H.W., Tucker J.M., Arribere J.A, & Garfinkel D.J. (2017). Ribosome Biogenesis Modulates Ty1 Copy Number Control in Saccharomyces cerevisiae. Genetics, 207(4), 1441-1456.

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Extracellular Vesicle Isolation and Characterization

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Extracellular vesicle (including exosomes and microvesicles) preparation and examination were performed according to previous description (Jung and Mun, 2018 ). LMH cells were cultured to near confluency in a 75 cm dish, and cells were mock infected or infected at a MOI of 1 in serum-free DMEM at 4 °C for 1 h. Then cells were washed once with PBS and cultured in extracellular vesicle-free full DMEM medium in the absence or in the present of PP1 or PP2. Medium was harvested after 24 hpi and extracellular vesicles were purified by differential centrifugation at 4 °C (300×g for 10 min, 2000×g for 20 min, 10000×g for 40 min using a Beckman JA-25.15 fixed angle rotor, then ultracentrifuged 100000×g for 90 min using a Beckman SW-41 swinging bucket rotor). The extracellular vesicle pellets generated were resuspended in serum-free DMEM, fixed and dried. Then samples were coated with gold/palladium alloy by sputter coating and examined under a Hitachi H-7650 transmission electron microscope (Hitachi High Technologies, Shanghai, China), and images were taken using an AMT CCD camera (Advanced Microscopy Techniques, Woburn, MA).

Wang Z., Sun B., Gao Q., Ma Y., Liang Y., Chen Z., Wu H., Cui L., Shao Y., Wei P., Li H, & Liu S. (2019). Host Src controls gallid alpha herpesvirus 1 intercellular spread in a cellular fatty acid metabolism-dependent manner. Virology, 537, 1-13.

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Purification of Rift Valley Fever Virus

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Vero cells were grown to 80% confluency in ten 175 cm² flasks and infected with RVFV virus at a multiplicity of infection (MOI) equal to 0.1. At 72 hours post-infection (p.i.), supernatants were harvested and cellular debris removed by centrifugation in a JLA 10.500 rotor (Beckman Coulter, Fullerton, CA) (4°C, 10 minutes, at 2600×g). Virus was precipitated from the clarified supernatant by adding polyethylene glycol (PEG) to a final concentration of 5% and stirring the solution overnight at 4°C. The precipitated virus was pelleted by centrifugation in the JLA 10.500 rotor (4°C, 30 minutes, at 2600×g), and the supernatants were decanted and viral pellets resuspended in a minimal (∼1 ml) volume of sterile PBS. Viral suspensions were applied to sterile 10–60% sucrose gradients and ultra-centrifuged for two hours at 32,000 rpm (126,000×g) in a SW-41 swinging bucket rotor (Beckman Coulter, Fullerton, CA). A distinct band corresponding to RVFV was removed and re-centrifuged overnight at 22,000 rpm in a JA 20 rotor (Beckman Coulter, Fullerton, CA) to pellet the virus. The supernatants were discarded and the RVFV was resuspended in a minimal volume of PBS, separated into aliquots and frozen at −80°C until analysis.

Nuss J.E., Kehn-Hall K., Benedict A., Costantino J., Ward M., Peyser B.D., Retterer C.J., Tressler L.E., Wanner L.M., McGovern H.F., Zaidi A., Anthony S.M., Kota K.P., Bavari S, & Hakami R.M. (2014). Multi-Faceted Proteomic Characterization of Host Protein Complement of Rift Valley Fever Virus Virions and Identification of Specific Heat Shock Proteins, Including HSP90, as Important Viral Host Factors. PLoS ONE, 9(5), e93483.

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Purification of Honeybee Virus Particles

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SBV and DWV were purified as described previously.[^12b, ²⁹] Briefly: one hundred experimentally infected honeybee pupae were homogenized using a Dounce homogenizer (piston‐wall distance 0.075 mm) in 50 mL of phosphate buffered saline (PBS) on ice. The extract was centrifuged at 15,000×g for 30 min at 10 °C. The pellet was discarded, and the supernatant was ultracentrifuged at 150,000×g for 3 h in a Ti50.2 fixed‐angle rotor (Beckman‐Coulter) at 10 °C. The resulting pellet was resuspended in PBS in a final volume of 10 mL. MgCl₂ was added to a final concentration of 5 mM as well as 20 μg/mL of DNase I and 20 μg/mL of RNase. The solution was incubated at room temperature for 30 min and centrifuged for 15 min at 5,500 g at room temperature. The resulting supernatant was loaded onto 0.6 g/mL CsCl in PBS and centrifuged for 16 h at 30,000 rpm in an SW41 swinging‐bucket rotor at 10 °C (Beckman‐Coulter). Virus bands were collected by the gentle piercing of ultracentrifuge tubes with an 18‐gauge needle. The viruses were buffer‐exchanged to PBS and concentrated using centrifuge filter units with a 100‐kDa molecular mass cutoff.

Šimonová A., Romanská V., Benoni B., Škubník K., Šmerdová L., Procházková M., Spustová K., Moravčík O., Gahurova L., Pačes J., Plevka P, & Cahová H. (2022). Honeybee Iflaviruses Pack Specific tRNA Fragments from Host Cells in Their Virions. Chembiochem, 23(17), e202200281.

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Isolation of Lipid-Rich Organelles from U-2 OS Cells

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10 15-cm plates of U-2 OS cells expressing inducible FSP1-GFP were induced with 10 ng/mL doxycycline for 48 hr. Cells were harvested by scraping into PBS and centrifuged for 10 min at 500 x g. Cell pellets were resuspended in cold hypotonic lysis medium (HLM, 20 mM Tris-HCl pH 7.4, 1 mM EDTA) supplemented with 1X cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich), incubated on ice for 10 min, dounced using 80X strokes and centrifuged at 1000 x g for 10 min. The supernatant was subsequently transferred to Ultra-Clear ultracentrifuge tubes (Beckman-Coulter), diluted with 60% sucrose/HLM to a final concentration of 20% sucrose/HLM, and overlaid by 4 ml of 5% sucrose/HLM followed by 4 ml of HLM. Overlaid samples were centrifuged for 30 min at 15,000 x g in an ultracentrifuge using a SW41 swinging bucket rotor (Beckman-Coulter). Buoyant fractions were collected using a tube slicer (Beckman-Coulter), additional fractions were pipetted from the top of the sucrose gradient in 1 mL increments, and pellets were resuspended in 1 mL HLM. 100 μL of 10% SDS was added to each fraction, yielding a final concentration of 1% SDS. Samples were then sonicated for 15 s and incubated for 10 min at 65°C. Buoyant fractions were incubated at 37°C for 1 hr and sonicated every 20 min, followed by a final incubation at 65°C for 10 min.

Bersuker K., Hendricks J., Li Z., Magtanong L., Ford B., Tang P.H., Roberts M.A., Tong B., Maimone T.J., Zoncu R., Bassik M.C., Nomura D.K., Dixon S.J, & Olzmann J.A. (2019). The CoQ oxidoreductase FSP1 acts in parallel to GPX4 to inhibit ferroptosis. Nature, 575(7784), 688-692.

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Honeybee Virus Purification Protocol

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Honeybee viruses were purified as described previously (13 (link), 16 (link), 52 (link)). Briefly, 50 experimentally infected honeybee pupae were homogenized with a Dounce homogenizer (piston-wall distance of 0.075 mm) in 30 ml of PBS [Dulbecco’s Phosphate-Buffered Saline Modified, D8537, Sigma-Aldrich; 2.7 mM KCl, 136.9 mM NaCl, 1.5 mM KH₂PO₄, and 8.1 mM Na₂HPO₄ (pH 7.4)] on ice. The extract was centrifuged at 15,000g for 30 min at 10°C. The pellet was discarded, and the supernatant was ultracentrifuged at 150,000g for 3 hours in a Ti50.2 fixed-angle rotor (Beckman-Coulter) at 10°C. The resulting pellet was resuspended in PBS in a final volume of 5 ml. MgCl₂ was added to a final concentration of 5 mM, as well as 20 μg/ml of deoxyribonuclease I and 20 μg/ml of RNase. The solution was incubated at room temperature for 30 min and centrifuged for 15 min at 5500g at room temperature. The resulting supernatant was separated using a CsCl (0.6 g/ml) gradient in PBS by ultracentrifugation for 16 hours at 30,000 rpm in an SW41 swinging-bucket rotor at 10°C (Beckman-Coulter). Virus bands were collected by the gentle piercing of ultracentrifuge tubes with an 18-gauge needle. The viruses were buffer exchanged to PBS and concentrated using centrifuge filter units with a 100-kDa molecular mass cutoff.

Škubník K., Sukeník L., Buchta D., Füzik T., Procházková M., Moravcová J., Šmerdová L., Přidal A., Vácha R, & Plevka P. (2021). Capsid opening enables genome release of iflaviruses. Science Advances, 7(1), eabd7130.

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Polysome Fractionation and RNA Extraction

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1×10⁷ cells were harvested using polysome extraction buffer (150 mM KOAc, 2.5 mM Mg(OAc)₂, 20 mM K-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, pH. 7.5, RNaseOUT, and protease inhibitor cocktail). Lysates were fractionated using a linear sucrose gradient (10-50% sucrose) in a Beckman Coulter SW41 swinging bucket rotor (35, 000 r.p.m., 3 hrs, 4 °C). Heavy fractions (#9, 10 and 11, see Fig.5k) were isolated for RNA extraction using Trizol.

Landon A.L., Muniandy P.A., Shetty A.C., Lehrmann E., Volpon L., Houng S., Zhang Y., Dai B., Peroutka R., Mazan-Mamczarz K., Steinhardt J., Mahurkar A., Becker K.G., Borden K.L, & Gartenhaus R.B. (2014). MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL. Nature Communications, 5, 5413.

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Lipid Raft Isolation from Membranes

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Lipid raft fractions were prepared using a method adapted from a prior report [29 (link)]. Brush border membrane extracts and BeWo plasma membranes were incubated in TNE buffer (25 mM Tris HCl, 150 mM NaCl, 5 mM EDTA), supplemented with 1% Lubrol or 1% Triton X-100, respectively, on ice for 1 h. The mixtures were then mixed with OptiPrep (60% iodixanol) to a final concentration of 40% iodixanol in a final volume of 3 mL. This was added to the bottom of an open-top ultra-centrifuge tube and overlayed with 6 mL of 30% iodixanol and then 2 mL of 5% iodixanol. Samples were centrifuged at 260,000 g for 4 h at 4°C using an SW41 swinging bucket rotor (Beckman Coulter). Detergent-resistant lipid rafts migrated up the tube and concentrated at the 30–5% interface. Ten 1.4 mL fractions were collected from each tube with a pipette, starting from the least dense fractions at the top of the tube.

Szilagyi J.T., Vetrano A.M., Laskin J.D, & Aleksunes L.M. (2017). Localization of the Placental BCRP/ABCG2 Transporter to Lipid Rafts: Role for Cholesterol in Mediating Efflux Activity. Placenta, 55, 29-36.

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Membrane Protein Purification and Characterization

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EPEC gfcD::kan / pSA10(GfcD) was grown overnight in 5 mL of LB broth containing 100 μg mL^–1 ampicillin. The following day, 100 mL of LB with 100 μg mL^–1 ampicillin was inoculated with 1 mL of overnight culture and grown until OD600 reached 0.6. IPTG (0.5 mM) was added to the cultures and kept at 30°C for an additional 7 hours with shaking. Lysis and harvesting of membranes were described above in Large scale expression of GfcD. Membrane pellets were re-suspended in 50 mM sodium phosphate pH 7.4, 10% glycerol, 20% sucrose, and 250 μL of the membrane solution was added to the top of a discontinuous gradient of layered solutions—4 mL of 70% sucrose, 4 mL of 60% sucrose, and 3.75 mL of 20% sucrose—in an ultraclear tube. The tubes were loaded in a Beckman SW-41 swinging bucket rotor and centrifuged at 28,000 rpm (134,000 g) for 16 hours at 4°C. At the end of the sedimentation, tubes were punctured at the bottom with a hot needle and 800 μL fractions were collected. Fractions were analyzed for NADH oxidase activity, run on SDS-PAGE and then blotted.

Larson M.R., Biddle K., Gorman A., Boutom S., Rosenshine I, & Saper M.A. (2021). Escherichia coli O127 group 4 capsule proteins assemble at the outer membrane. PLoS ONE, 16(11), e0259900.

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