S. acidocaldarius exosomes were isolated from the supernatant obtained after cell growth. The procedure was adapted from Ellen et al., 2009 (link). The supernatant was split into 8 fractions and exosomes were pelleted in two runs of ultracentrifugation (Optima LE-80K, Beckman Coulter) at 125,000 × g for 45 min at 4°C. The pellet was resuspended in 2 ml (per fraction) of the supernatant and ultracentrifuged (Optima MAX-TL, Beckman Coulter) at 12,000 rpm (TLA55 rotor, Beckman Coulter) for 10 min at 4°C. The pellet (containing intact cells and cell debris) was discarded, and the supernatant was ultracentrifuged (Optima MAX-TL, Beckman Coulter) at 42,000 rpm (TLA55 rotor, Beckman Coulter) for 90 min at 4°C. The pellet containing the isolated exosomes was resuspended in MilliQ water at a concentration of 15 mg/ml. The purity of the sample was assessed by negative staining with 1% uranyl acetate on 300 mesh Quantifoil copper grids with continuous carbon film (EM Resolutions).
Tla 55 rotor
Manufactured by Beckman Coulter
Sourced in United States, Germany
The TLA-55 rotor is a high-speed fixed-angle centrifuge rotor designed for use with Beckman Coulter ultracentrifuges. It is capable of reaching maximum speeds of up to 100,000 rpm, generating forces up to 436,000 x g. The TLA-55 rotor is commonly used for a variety of applications that require high-speed centrifugation, such as the separation and isolation of biological macromolecules, cell organelles, and other small particles.
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Lab products found in correlation
Sw32ti rotor, by Beckman Coulter (6 mentions)
Optima max xp, by Beckman Coulter (5 mentions)
Sw55ti rotor, by Beckman Coulter (5 mentions)
Sw28 rotor, by Beckman Coulter (4 mentions)
Optima tlx ultracentrifuge, by Beckman Coulter (3 mentions)
Optiprep, by Merck Group (3 mentions)
Ti45 rotor, by Beckman Coulter (2 mentions)
Sw56 rotor, by Beckman Coulter (2 mentions)
Optima max tl, by Beckman Coulter (2 mentions)
Fl winlab software version 4, by PerkinElmer (2 mentions)
Nanosight ns300 nanoparticle tracking analysis system, by Malvern Panalytical (2 mentions)
Optima max ultracentrifuge, by Beckman Coulter (2 mentions)
Sw41ti rotor, by Beckman Coulter (2 mentions)
Paclitaxel, by Beckman Coulter (2 mentions)
Biorupter ucd 300 bath sonicator, by Diagenode (1 mentions)
Ody 2816 imager, by LI COR (1 mentions)
Chloroform methanol, by Merck Group (1 mentions)
Atto 550, by ATTO-TEC (1 mentions)
Sw55 rotor, by Beckman Coulter (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
93 protocols using tla 55 rotor
1
Isolation of S. acidocaldarius Exosomes
2
Solid-State NMR Sample Preparation
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For the preparation of solid-state NMR samples lyophilized tubulin (Cytoskeleton, Inc.) was solubilised in BRB80 buffer (80 mM PIPES, 2 mM MgCl2, 1 mM EGTA, pH 6.8, 1 mM NaN3, 1 mM DTT, pH 6.8 supplemented with protease inhibitor (Sigma-Aldrich, cOmplete EDTA-free), to a final concentration of 2 mg/mL). Then 1 mM Guanosine-5’-triphosphate (GTP) was added and incubation took place for 15 min at 30 °C. In the following, 20 μM paclitaxel (Taxol, SIGMA) was used to stabilize the MT and incubation took place for another 15 min at 30 °C. The MT were spun down at 180,000 g (Beckman TLA-55 rotor) for 30 min at 30 °C and the pellet was resuspended in warm BRB80 buffer with 20 μM paclitaxel. Subsequently 0.55 mg/mL [13C-15N]-MAP7 MTBD was added. The interaction partners were incubated for 30 min at 30 °C. In the following step [13C-15N]-MAP7 MTBD in complex with MT was separated from the unbound, non-polymerised fraction by centrifugation at 180.000 g (Beckman TLA-55 rotor) for 30 min at 30 °C. Afterwards, the pellet was washed with BRB80 buffer containing protease inhibitor, without disturbing the pellet. A 1.3 mm rotor was packed with the pellet.
3
Purification and Characterization of Virus-Like Particles
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Culture media from transfected cells containing VLPs was collected and centrifuged twice at 200 g for 10 min at room temperature. Supernatants were added to ultracentrifuge tubes with a 20% sucrose/Tris-buffer saline (TBS 20 mM Tris, pH approx. 7.4, 0.9% NaCl) cushion, followed by ultracentrifugation at 151.000 g for 2 h at 4°C in a SW41TI rotor (Beckman Coulter). Pellets, containing the VLPs, were resuspended in TBS buffer and ultracentrifuged at 112.000 g for 15 min at 4°C in a TLA55 rotor (Beckman Coulter) in the 20% sucrose/TBS cushion. Pellets containing the VLPs were collected.
To test the effect of CPC (Merck), purified VLPs were incubated with CPC (from 0.5% to 0.0005%) or Sodium dodecyl sulfate (SDS) (0.5%) for 1 min, both detergents dissolved in PBS (Both detergents were obtained as pure powder from Merck, USA). Samples were ultracentrifuged at 112.000 g for 15 min at 4°C in a TLA55 rotor (Beckman Coulter) in a 20% sucrose/TBS cushion. The supernatant and pellet were recovered and analysed by Western blot.
For electron microscopy visualisation, the VLPs collected from the medium were filtered using a 0.2 µm filter before the centrifugations and the TBS buffer was exchanged for TNE buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA, pH 7.4).
To test the effect of CPC (Merck), purified VLPs were incubated with CPC (from 0.5% to 0.0005%) or Sodium dodecyl sulfate (SDS) (0.5%) for 1 min, both detergents dissolved in PBS (Both detergents were obtained as pure powder from Merck, USA). Samples were ultracentrifuged at 112.000 g for 15 min at 4°C in a TLA55 rotor (Beckman Coulter) in a 20% sucrose/TBS cushion. The supernatant and pellet were recovered and analysed by Western blot.
For electron microscopy visualisation, the VLPs collected from the medium were filtered using a 0.2 µm filter before the centrifugations and the TBS buffer was exchanged for TNE buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA, pH 7.4).
4
Purification of Extracellular Vesicles Using Iodixanol Gradient
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Density gradient ultracentrifugation was performed as described previously with modifications (Kalra et al., 2013 (link)). A discontinuous iodixanol gradient consisting of 40% w/v, 20% w/v, 10% w/v, and 5% w/v solutions of iodixanol was prepared by diluting a stock solution of OptiPrep [60% w/v aqueous iodixanol (Sigma)] in 0.25 M sucrose/10 mM Tris, pH 7.5. The crude EVs isolated from the F. oxysporum f. sp. vasinfectum culture by differential centrifugation coupled with ultracentrifugation, were resuspended in the OptiPrep solution, and overlaid onto the top layer. A control tube consisting 3 ml of each 40%, 20%, 10%, and 5% solutions was also prepared. Both tubes were simultaneously subjected to ultracentrifugation at 100,000 × g for 18 h at 4°C (Beckman Coulter: SW-28 rotor). The 12 fractions were collected separately for further analysis. EVs were pelleted by ultracentrifugation at 100,000 × g for 1 h at 4°C (Beckman Coulter: TLA-55 rotor). Pellets were then washed with 1 ml of PBS and the supernatant was removed with two successive ultracentrifugations at 100,000 × g for 1 h at 4°C (Beckman Coulter: TLA-55 rotor) and resuspended in 30 µl before being stored at -80°C.
5
Amyloid Seeding Assay for Synucleinopathies
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LB α-Syn from brain extracts of patients pathologically confirmed with either AD or PDD were sonicated on high for 20 cycles of 30 s on, followed by 30 s off (Diagenode Biorupter UCD-300 bath sonicator). The amplification reaction was set up with 5% LB α-Syn (calculated based on sandwich ELISA) and 95% α-Syn monomer and incubated in DPBS (pH 7.4, without Mg2+ and Ca2+) at 37 °C with constant agitation at 1000 rpm. The samples were incubated for 4–14 days in a 50–100 μl volume and the resulting samples were termed passage 1 (P1). Amplification progress was determined by removing a 2 μl aliquot and diluting it tenfold in DPBS to a final α-Syn concentration of 10 ng/μl. Samples were centrifuged at 100,000 ×g for 30 min at 24 °C in a TLA-55 rotor (Beckman Coulter). The supernatants were carefully removed, and the pellets were suspended in 20 µl of DPBS. Equal volumes of supernatant and pellet fractions were analyzed on SDS-PAGE gels and Western blot as above. After blocking, the membranes were probed with rabbit anti-α-syn antibody (HuA) and IRDye- labeled secondary antibodies and scanned using ODY-2816 Imager (Li-Cor Biosciences).
6
Characterization of Mitochondrial Protein Cox26
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Analyses were performed essentially as previously described [26] (link). For membrane-association analysis isolated mitochondria containing Cox26 FLAG were subjected to extraction in 0.1 M Na 2 CO 3 (pH 10.8), or lysed with 0.1% Triton X-100 supplemented with 0.4 M KCl. Membranes and soluble fractions were separated by centrifugation for 1 h at 100,000 rpm, 4 °C in a TLA-55 rotor (Beckmann). Samples were precipitated with trichloroacetic acid (TCA) and analyzed by SDS-PAGE and Western-blotting. For protease protection assays mitochondria were resuspended in iso-osmotic SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2), converted to mitoplasts by hypotonic swelling in EM buffer (1 mM EDTA, 10 mM MOPS, pH 7.2), or lysed in 0.2% Triton X-100 for 20 min on ice and subsequently treated with indicated amounts of proteinase K (PK) for 10 min on ice. Samples were precipitated with TCA and analyzed by SDS-PAGE and Western-blotting.
7
Protein Extraction from Mouse Lung
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For protein extraction, the mouse lung tissue was homogenized in a buffer containing 20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM PMSF and protease inhibitors, using a Brinkman Polytron homogenizer (Brinkman, Oxford, CT, United States). Total lysates were prepared by adding SDS and Triton X-100 to a final concentration of 0.3 and 1%, respectively, with gently mixing for 2 h at 4°C. The resulting lysates were clarified by centrifugation (191,500 ×g, 45 min, 4°C) in a Beckman Optima Max-XP ultracentrifuge with a TLA-55 rotor (Beckman, Indianapolis, IN, United States). Protein concentration was determined by the micro BCA method with BSA as standard.
8
Norovirus Virus-Like Particle Production
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Full-length VP1 genes for GI.1 West Chester, GII.4 Saga 2006, GII.10 Vietnam, GII.17 Kawasaki308, and GII.17 Saitama T87 (GenBank accession numbers: AY502016.1, AB447457.1, AF504671.2, LC037415.1, AII73747.1) were cloned and expressed in a baculovirus system [29 (link),30 (link)]. After transfection of a bacmid containing the recombinant VP1 gene in Sf9 insect cells and incubation for 5–7 days, the culture medium was collected and centrifuged for 10 min at 3000 rpm at 4 °C. Subsequently, Hi5 insect cells were infected with recovered baculovirus and incubated for 5 days. After centrifuging the culture medium for 10 min at 3000 rpm at 4 °C and then 1 h at 6500 rpm at 4 °C, VLPs in the supernatant were concentrated by ultracentrifugation at 35,000 rpm (Beckman Ti45 rotor, Krefeld, Germany) for 2 h at 4 °C. Furthermore, VLPs were further purified using CsCl equilibrium gradient ultracentrifugation at 35,000 rpm (Beckman SW56 rotor, Krefeld, Germany) for 18 h at 4 °C. VLPs were pelleted for 2 h at 40,000 rpm (Beckman TLA55 rotor, Krefeld, Germany) at 4 °C and solved in PBS (pH 7.4).
9
Purification and Lipid Analysis of Podocyte Membranes
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The podocyte cell membranes were purified as previously described [28 (link)]. Briefly, PON2-proficient and -deficient cells were lysed in a hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.05% Nonidet P-40, pH 7.9), homogenized in a Dounce glass/glass homogenizer, and subjected to a sequential centrifugation. Nuclei and mitochondria were segregated from the sample by a centrifugation at 15 × 103× g, and membrane fractions [endoplasmatic reticulum (ER) and plasma membrane] were collected as the pellet after a final ultracentrifugation step at 105× g in a Beckman TLA 55 rotor (Beckman Coulter, Fullerton, CA, USA). The membrane pellets were resuspended in water and the lipids were extracted using chloroform/methanol 2:1 (v/v) (Sigma-Aldrich) and internal standards (67 pmol PC 17:0–14:1, 81 pmol PE 17:0–14:1, 63 pmol PI 17:0–14:1, 68 pmol PS 17:0–14:1, 77 pmol PG 17:0–14:1, 82 pmol PA 17:0–14:1 (Avanti Polar Lipids)). The sample was sonicated and vigorously vortexed and the phase separation was induced by a centrifugation (800× g for 5 min at 4 °C). After repetitive extractions, the organic phases were combined and dried under a stream of nitrogen. The dried lipid extracts were analyzed by LC-ESI-MS/MS as previously described.
10
Preparation and Tethering of GUVs
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GUVs were prepared as described (Malsam et al., 2012 (link)), with some modifications. A 1 µmol amount of dried lipid mix (68.5 mol% POPC, 18 mol% POPE, 8 mol% ergosterol, 1 mol% DAG, 1 mol% PI-3-P, 0.5 mol% Atto550 [AttoTec], and 3% DOGS-NTA [or compensatory amounts of POPC]) was resolved in trehalose buffer (20 mM trehalose, 1 mM HEPES/KOH, pH 7.4, 1% glycerol [vol/vol], 1 mM DTT) for a final lipid concentration of 2 mM. Lipids were then sedimented by a centrifugation step in TLA-55 rotor (Beckman) for 2 h at 55,000 rpm. Pellets were resuspended in a total volume of 20 µl and spread as a uniform layer (diameter, ∼10 mm) on the surface of ITO-coated glass slides (Nanion Technologies). The lipid suspension was dried for 12 h at low vacuum (100 mbar). An O-ring (20 × 2 mm) between two ITO slides formed the electroformation chamber, which was filled with GUV swelling buffer (240 mM sucrose, 1 mM HEPES/KOH, pH 7.4, 1 mM DTT). Electroformation was carried out for 15 h at 10 Hz and 1 V at room temperature. GUVs were collected from the slides by gentle pipetting and were ready to use for tethering assays.
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