At passage 6 and at ~80% confluency, cells were primed with 1500 UI/mL IFN-β (Merck Millipore, Ireland) for 1 hour in hPL-free DMEM as previously described.41 (link) Primed with IFN-β and non-primed MSCs were washed with PBS and cultured for an additional 48 hours in media containing EVs-depleted hPL (hPL was EV depleted by ultracentrifugation at 120 000 × g for 18 hours at 4°C; Ti70 rotor, κ-factor 298.0, Beckman Coulter, Ireland), and the CM was collected. To remove cells, CM (100 mL) was centrifuged at 2000 × g for 10 minutes. To remove debris and large-sized EVs, supernatants were centrifuged at 10 000 × g for 45 minutes at 4°C in a Sorvall ST 8 small benchtop centrifuge using an HIGHConic III fixed angle rotor (κ-factor 2488, Thermo Fisher Scientific, Ireland), followed by filtration using a 0.2-µm pore filter membrane (Thermo Fisher Scientific). Next, small-sized EVs were isolated using the polyethylene glycol 6000 (PEG) precipitation protocol as previously described43-45 (link) followed by ultracentrifugation at 100 000 × g for 91 minutes (Ti70 rotor, κ-factor 298.0, Beckman Coulter, Ireland) at 4°C. MSC-EV samples were resuspended in 0.5 mL of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/0.9% sodium chloride (NaCl, Thermo Fisher Scientific) and stored at−80°C.
Ti70 rotor
Manufactured by Beckman Coulter
Sourced in United States, Ireland
The Ti70 rotor is a centrifuge rotor designed for high-speed applications in the laboratory. It is capable of achieving a maximum speed of 70,000 rpm and can generate a maximum RCF (Relative Centrifugal Force) of 500,000 x g. The rotor is compatible with a variety of tubes and sample containers, allowing for efficient separation and processing of various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Spin x centrifuge tube filter, by Corning (2 mentions)
Sw32ti rotor, by Beckman Coulter (2 mentions)
Whatman filter, by GE Healthcare (2 mentions)
Zetaview nanoparticle tracking analysis system, by Particle Metrix (2 mentions)
Ab219209, by Abcam (2 mentions)
Ab125011, by Abcam (2 mentions)
Ab13504, by Abcam (2 mentions)
Ab254175, by Abcam (2 mentions)
Nanosight ns300 system, by Malvern Panalytical (2 mentions)
Nalgene filter, by Thermo Fisher Scientific (2 mentions)
Wallac envision 2104 multilabel reader, by PerkinElmer (2 mentions)
Optima le 80k, by Beckman Coulter (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Exo fect exosome transfection reagent, by System Biosciences (2 mentions)
Fetal bovine serum (fbs), by System Biosciences (2 mentions)
Emulsifex, by Avestin (1 mentions)
Steritop filtration unit, by Merck Group (1 mentions)
Vivaflow 200, by Sartorius (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
59 protocols using ti70 rotor
1
Isolation of Mesenchymal Stem Cell-Derived Extracellular Vesicles
2
Ribosome Purification for Translation Assays
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Ribosomes were purified from the strains HDB140, HDB143, and HDB144. The strains were cultured in Lysogeny broth (LB) to an A600 of 1.0 at 37°C and chilled on ice for 15 min before they were harvested by centrifugation at 4000 g for 10 min. The cell pellet was washed twice with Buffer A at pH 7.5 (10 mM Tris-OAc, 14 mM Mg(OAc)2, 60 mM KOAc, 1 mM DTT, 0.1% Complete Protease Inhibitor) and lysed using the Emulsifex (Avestin) at a pressure of 8000 psi. The cell lysate was loaded on a sucrose cushion at pH 7.5 (50 mM Tris-OAc, 1 M KOAc, 15 mM Mg(OAc)2,1.44 M sucrose, 1 mM DTT, 0.1% Complete Protease Inhibitor) and centrifuged at 80,000xg in a Ti70 rotor (Beckman-Coulter) for 17 hr. The obtained ribosomal pellet was resuspended in Buffer B at pH 7.5 (50 mM Tris-OAc, 50 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT), flask frozen in liquid nitrogen and stored at −80°C. This suspension of ribosomes is presumed to consist of a pool of non-translating 30S, 50S and 70S particles due to the concentration of Mg2+ in the buffer they are in. Each batch of ribosomes that was prepared was tested for optimal translation by titrating different volumes in the PURExpress Δ-Ribosome kit.
3
Optimized OMV Extraction from B. thetaiotaomicron
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In order to obtain a sufficient quantity of OMVs to detect a metabolomic signal, 2 l of RM (three culture flasks grown independently) and DM (three culture flasks grown independently) were inoculated with overnight culture of B. thetaiotaomicron at an initial OD of 0.0005 and 0.05, respectively. The OMV extraction was performed as previously described (Stentz et al., 2014 (link)) with slight modifications. After 16 h (OD approximately 4.0), the cell cultures were rapidly cooled in a manually shaken ice bath. The cultures were then centrifuged at 5000 × g for 15 min at 4°C, and the supernatants filtered through a 0.22-μm Steritop filtration unit (Millipore, Billerica, MA, United States) to remove debris and cells. The sterility of the filtrate containing the vesicles was confirmed by plating onto BHI–hemin agar. OMVs in the 2 l filtrates were concentrated by molecular weight (100 kDa MWCO, Vivaflow 200, Sartorius) down to 2 ml, diluted by addition of 1 l of ice-cold PBS, pH 7.4, and the suspensions were filtered and concentrated again, down to 9 ml. The 9 ml retentate was ultracentrifuged [150,000 × g for 2 h at 4°C in a Ti70 rotor (Beckman Instruments)]. The supernatant was completely removed using a vacuum pump and a needle and the OMV pellets were snap frozen in liquid nitrogen and stored at -80°C before extraction.
4
Exosome Isolation from Cell Cultures
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CEM and HUT102 cells were grown in complete media supplemented with 10% exosome-free FBS, and exosomes were isolated from 500 mL of cell culture grown in a roller bottle over the course of four weeks. Cells were pelleted by centrifugation at 1000× g for 10 min, and the cell supernatant was collected. An additional centrifugation at 2000× g for 10 min was used to pellet dead cells and cell debris. The supernatant was collected and ultracentrifugation in a Ti70 rotor (Beckman Coulter; Indianapolis, IN, USA) was performed at 2000× g for 45 min, 10,000× g for 45 min, 100,000× g for 90 min, and 167,000× g for 16 h to pellet EVs to obtain 2K, 10K, 100K, and 167K EV populations, respectively. For total EVs, a 100,000× g spin was performed for 90 min to pellet all EVs. All pellets were then re-suspended in Dulbecco’s phosphate-buffered saline without calcium and magnesium (PBS), consolidated into a single tube per each EV population, and washed with PBS. The resulting pellet was re-suspended in 300 µL of PBS. All centrifugations were performed at 4 °C.
5
Isolation of Extracellular Vesicles from BMSC
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EVs were prepared from the supernatant of BMSCs by differential centrifugation according to current protocols28 (link). Briefly, BMSC-conditioned medium cultured for 24 hours was harvested, centrifuged at 500 g for 30 min to eliminate cells and at 16 500 g for 20 min, followed by filtration through a 0.22 μm filter to remove cell debris. EVs were pelleted by ultracentrifugation (Beckman Ti70 rotor, USA) at 120 000 g for 120 min. EVs were measured for their protein content using a BCA Protein Assay Kit (Pierce, USA).
6
Adeno-Associated Virus Production Protocol
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Purified AAVs were produced as previously described (Uezu et al., 2016 (link)). Briefly, HEK293T cells grown on 150mm dishes were transfected with GEARBOCS plasmid, helper plasmid pAd-DeltaF6 and the serotype plasmid AAV PHP.eB. After three days, cell lysates were prepared with 15mM NaCl, 5mM Tris-HCl, pH 8.5 followed by three repeats of freeze-thaw cycles. The cell lysates were centrifuged for 30min at 4000rpm to collect the supernatant. Benzonase-treated supernatant (50U/ml, 30 min at 37°C) was added to an Optiprep density gradient (15%, 25%, 40% and 60%) for ultracentrifugation at 60,000 rpm for 1.5hr using a Beckman Ti-70 rotor. AAV fraction was collected from the gradient and concentrated along with the multiple washes with DPBS in a 100 kDa filtration unit. AAV titers were quantified by qPCR based on SYBR green technology using primer pair targeting AAV2 ITR (Aurnhammer et al., 2012 (link)).
7
Purification of S. cerevisiae Ribosomes
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S. cerevisiae G2400A mutant 80S ribosomes were purified for the structural studies according to the published protocol73 (link). The ribosomes for the drug binding assays were purified using the same protocol except that sucrose gradient fractionation was replaced with pelleting through a 30% sucrose cushion in a buffer containing 20 mM Hepes-KOH, pH 7.5, 120 mM KCl, 8.3 mM MgCl2, 2 mM DTT, 0.3 mM EDTA. Specifically, ribosomes collected from the lysate by sequential differential precipitation with PEG 20 K (4% and then 9% w/v) were resuspended in 15 mL of buffer A (30 mM Hepes-KOH, pH 7.5, 150 mM KCl, 10 mM MgCl2, 8.5% mannitol, 2 mM DTT, 0.5 mM EDTA), layered over a 15 mL sucrose cushion, and centrifuged for 16 h in a Ti-70 rotor (Beckman) at 36,000 rpm (130,000×g) at 4 °C. The ribosome pellets were resuspended in a storage buffer containing 10 mM Hepes-KOH, pH 7.5, 50 mM KOAc, 10 mM NH4Cl, 2 mM DTT, 5 mM Mg(OAc)2, flash frozen in liquid nitrogen and stored at −80 °C.
8
Isolation of Membrane Proteins from Transfected Cells
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Transfected cells were collected, re-suspended in ice-cold hypotonic buffer (10 mmol/L Tris-HCl pH 8.0, 10 mmol/L KCl, 5 mmol/L EDTA) containing protease inhibitor cocktail (Roche) and incubated for 30 min at 4 °C on a rotator. All subsequent steps were carried out at 4 °C. Cells were disrupted with ten strokes using a tight-fitting Potter-Elvehjem homogenizer. Cellular debris was spun down for 10 min at 400 × g and the supernatant was further centrifuged at 1000 × g for 10 min. The resulting supernatant was centrifuged at 265000 × g in a Ti70 rotor (Beckman). The membrane pellet was re-suspended in 50 mmol/L Hepes-Tris, pH 7.4, 100 mmol/L KNO3, 50 mmol/L sucrose, snap-frozen in liquid nitrogen and stored at -80 °C until further use.
9
Isolation and Characterization of Extracellular Vesicles
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EVs from biopsy culture cells were obtained from conditioned media of 107 cells at 80% of confluence, and grown with 10% Exo-FBS for 24 h (FBS depleted of exosomes, SBI, System Bioscience). Plasma EVs from tumor patients were isolated from 10 ml of blood collected during surgery or from 10 ml of blood from healthy donors. Conditioned media and plasmas were once centrifugated for 10 min at 4 °C and 400 × g, followed by three times at 5000 × g. The supernatant was then ultracentrifugated twice at 14,000 × g for 35 min at 4 °C in a Beckman ultracentrifuge with Ti70 rotor [21 (link)]. The resulting pellets were resuspended in PBS with 3.2% NaCitrate (0.11 M) and 1× protease inhibitor cocktail (Sigma-Aldrich) and stored at −80 °C. Equal amounts of MV proteins (quantified by Bradford assay) were used in all functional assays.
10
Lentiviral Vector Production and Purification
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The full-length nucleotide sequence of EPB41L4A-AS2 (NR_027706.1) was directly synthesized by Sangon Biotech Company and cloned into pLVX-Puro lentiviral expression vector (Clontech) between EcoRI and XbaI sites using an In-Fusion Cloning kit (Clontech). Lentiviruses were produced in 293T cells. Briefly, 293T cells were transiently transfected with pLVX plasmid and the packaging plasmids pLP1, pLP2, and pLP/VSVG using lipofectamine 2000. Lentiviral particles were harvested by collecting the supernatants 72 h later, which were centrifuged at 1500 g for 5 min and filtered through a 0.45 μm filter to remove cellular debris. Then, the crude lentivirus was concentrated by ultracentrifugation at 22,000 rpm for 2.5 h at 4°C, using a Beckman Ti70 rotor. The pellets were resuspended in DPBS and incubated at 4°C overnight. The purified lentivirus was then stored at −80°C.
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