Breast tissue at fifth, sixth, and seventh week was homogenized in PBS pH 7.4 in the presence of a protease inhibitor. The homogenate was centrifuged at 14 000 G for 15 min. The supernatant was transferred to a polycarbonate tube and centrifuged in a 90Ti rotor (Beckman, Brea, CA, USA) at 100,000 G for 1 h at 4 °C. The pellet was resuspended in lysis buffer for 2D Electrophoresis: 6 M urea, 50 mMDTT, 2% CHAPS in the presence of a protease inhibitors cocktail (Thermo Scientific, Waltham, MA, USA).
90 ti rotor
Manufactured by Beckman Coulter
Sourced in United States, Canada, Switzerland
The 90 Ti rotor is a high-speed centrifuge rotor designed for Beckman Coulter ultracentrifuges. It is capable of reaching speeds of up to 90,000 revolutions per minute (RPM) and can achieve relative centrifugal forces (RCF) of up to 436,000 x g. The rotor is made of titanium, which provides strength and durability for high-speed centrifugation applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
Avanti j e centrifuge, by Beckman Coulter (1 mentions)
Vacuum manifold, by Qiagen (1 mentions)
Microcentrifuge 5424 r, by Eppendorf (1 mentions)
Sx0002500, by Merck Group (1 mentions)
Nano zs instrument, by Malvern Panalytical (1 mentions)
Amicon centrifugal filter, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
45 ti rotor, by Beckman Coulter (1 mentions)
12 protocols using 90 ti rotor
1
Breast Tissue Fractionation for Proteomics
2
Exosome Isolation and Characterization
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Exosomes were obtained after 300×g for 5mins, 3,000×g for 20 mins, 6,000×g for 40 mins and 10,000×g for 60 mins. Cell-free supernatants were ultracentrifuged at 100,000×g for 1h at 4°C (Beckman 90 Ti rotor). After washed by PBS, exosomes submitted to a second ultracentrifugation in the same conditions. Then quantify the protein content of exosomes by BCA protein assay kit (PIERCE, Rockford, IL, USA). Eosomes were stored at -80°C.
3
Extracellular Vesicle Isolation Protocol
4
Isolation of Extracellular Vesicles from 4T1 Cells
5
Isolation and Characterization of Outer Membrane Vesicles
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OMVs were obtained from bacterial culture supernatants as described previously (Vanaja et al., 2016 (link)). Briefly, bacterial cultures were grown in LB broth at 37°C for 8 h to achieve abundant OMV production. Subsequently, supernatants of BL21/pEmpty or BL21/pMCR‐1 at early stationary phase were obtained by centrifuging and filtering to remove residual cells. The sterile filtrates were subsequently concentrated by ultrafiltration using a 100‐kDa Amicon centrifugal filters (Millipore), and the collected retentates were transferred to ultracentrifuge tubes and ultracentrifuged at 150,000 g for 3 h using a 90 Ti rotor (Beckman). The vesicle‐free supernatant fraction was collected, and the vesicle pellet was washed and resuspended in sterile PBS for use or stored at −80°C for further analysis. Each OMV sample was visualized by transmission electron microscope (TEM) according to standard procedures, of which particle size distribution and zeta potential were determined using a Nano ZS instrument (Malvern), the protein and LPS contents were quantified by BCA assay and Limulus Amebocyte lysate assay, respectively. The protein and LPS productions of OMVs were normalized by dividing by corresponding OD600nm value of each culture per millilitre, and these values were further divided by the OMV production of BL21/pEmpty to give rise to relative folds of OMV productions.
6
Outer Membrane Vesicles of V. fischeri
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Outer membrane vesicles (OMVs) produced by V. fischeri ES114 were prepared as previously described (61 (link), 62 (link)), with the following modification. After OMVs were separated from other extracellular products by ultracentrifugation, they were further purified by using linear 10 to 50% sucrose gradient centrifugation at 100 000 × g for 16 h at 4°C in a 90 Ti rotor (Beckman Coulter, Inc., Brea, CA). To treat squid with OMVs, 100 μg·ml−1 was added to FSW and incubated with hatchling animals for 24 h (61 (link), 62 (link)). The peptidoglycan monomer (tracheal cytotoxin [TCT]) and lipid A from V. fischeri were added to FSW for 24 h at a concentration of 1 μM and 10 μg·ml−1, respectively (25 (link), 26 (link)).
7
Membrane Protein Extraction from Heart Tissue
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Flash-frozen hearts were ground to a fine powder in liquid nitrogen using a pestle and mortar. Samples were homogenized on ice with 30 strokes of a glass-glass homogenizer, followed by 30 strokes in a Dounce homogenizer in (in mM) 250 sucrose, 1 EDTA, 10 HEPES, 1 DTT and pH 7.4 supplemented with protease inhibitor cocktail (Roche Applied Science). Following brief centrifugation (1000 g for 5 min at 4°C), the pellet was re-homogenized in fresh homogenization buffer with 25 strokes of a tight-fitting Dounce and cleared by brief centrifugation (1000 g, 5 min, 4°C). The resulting supernatant was combined with that of the previous step. The supernatant was centrifuged at 50,000 rpm using a 90 Ti rotor (Beckman Coulter, Brea, CA) for 1 hr at 4°C. The resulting membrane pellets were solubilized with rotation overnight at 4°C in 20 mM HEPES, 0.5% Triton X100, pH 7.4.
8
Exosome Isolation by Differential Centrifugation
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Exosomes were obtained as previously described [40 (link), 51 (link), 52 (link)]. Exosomes were collected by differential centrifugation and centrifuged at 300 × g for 5 minutes, 3,000 × g for 20 minutes, 6,000 × g for 40 minutes, 10,000 × g for 60 minutes, cell-free supernatants were ultracentrifuged at 100,000 × g for 1 h at 4°C (Beckman 90 Ti rotor). Exosomes were washed once with exosome-depleted PBS and submitted to a second ultracentrifugation in the same conditions. To quantify the protein content, exosomes pellets were suspended in exosome-depleted PBS and estimated by a BCA protein assay kit (PIERCE, Rockford, IL, USA) and exosomes were stored at −80°C and used within 5 days after isolation.
9
Phage Purification by CsCl Gradient
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A liter of filtered phage lysate (108–109 PFU/mL) was treated with 10 µg/mL of DNase I and RNase A (Sigma Aldrich Canada, Oakville, ON, Canada) and incubated at room temperature with gentle shaking for 1 h. After centrifugation at 7000× g at 4 °C for 18 h, the pellets were resuspended in 5–10 mL SM buffer (100 mM NaCl, 8 mM MgSO4•H2O, 50 mM 1 M Tris-Cl pH 7.5) at 4 °C for 3–4 h. The collected SM buffer was centrifuged at 12,000× g at 4 °C for 10 min and the supernatant was transferred to a fresh conical tube. Cesium chloride was gradually added to the supernatant, in four portions, until the final density of the mixture reached 0.817 g/mL. The phage-CsCl mixture was centrifuged in OptiSeal tubes (Beckman-Coulter Canada, Mississauga, ON, Canada) at 155,000× g at 4 °C for 24 h (90Ti rotor, Beckman-Coulter). The resultant visible band was extracted using a 20 g needle and syringe (BD Canada) and added to a fresh preparation of CsCl in SM buffer. The centrifugation and extraction process was repeated prior to an overnight dialysis using 3500 MWCO Slide-A-Lyzer dialysis cassettes (Thermo Scientific) against 1% (w/v) ammonium bicarbonate at 4 °C. Dialysis was repeated.
10
Isolation of Microvesicles from HEK 293 Cells
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HEK 293 cells (107) were gently washed with PBS. Microvesicle shedding was induced by treatment with PLY (2 µg/ml) in Ca-Tyrode’s buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH7.4, 2.5 mM CaCl2). The cells were incubated for 45 min at room temperature. Shed microvesicles were isolated according to a protocol for extracellular vesicles (28 (link)). Briefly, conditioned medium was centrifuged at 300 × g for 10 min at 4°C to pellet cells. Supernatant was centrifuged at 2,000 × g for 20 min at 4°C (2 k pellet), transferred to new tubes, and centrifuged in a 45 Ti rotor for 40 min at 10,000 × g (10 k pellet), and finally in a 90 Ti rotor (Beckman Coulter, Nyon, Switzerland) for 90 min at 100,000 × g (100 k pellet). The pellets were re-suspended in Ca-Tyrode’s buffer for stimulation of macrophages.
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