Exosomes were isolated according to the method described previously in our laboratory.7 (link) Serum (1 mL) was diluted with an equal volume of phosphate buffered saline (PBS; pH 7.4). Exosomes were isolated and purified by differential ultracentrifugation using a 30% sucrose cushion. In brief, to remove cells, centrifugation was initially performed at 2000 g at 4°C for 30 min, followed by 12,000 g at 4°C for 45 min. To remove the remaining debris, the supernatant was transferred to other centrifuge tubes and centrifuged at 110 000 g at 4°C for 120 min (Optima™ MAX-XP Ultracentrifuge, fixed angle MLA-55 rotor, Beckman Coulter Inc., Brea, CA, USA). To remove particles that were larger than 200 nm, the pellet was suspended in 2 mL of PBS and filtered through a 0.20 μm pore filter (Cellulose acetate, GVS™, Europe). The filtrate was centrifuged at 110 000 g at 4°C for 70 min following which the pellet was re-suspended in 2 mL PBS (pH 7.4) and centrifuged at 110 000 g for 70 min at 4°C. To purify the exosomes, the exosome pellet was suspended in 2 mL of PBS and subsequently purified using a 30% sucrose cushion and centrifuged at 110 000 g at 4°C for 75 min. The final pellet was re-suspended in 300 μL of PBS and stored at −80°C.
Mla 55 rotor
Manufactured by Beckman Coulter
Sourced in United States
The MLA-55 rotor is a fixed-angle, high-speed, micro-centrifuge rotor designed for Beckman Coulter laboratory equipment. It is capable of reaching a maximum speed of 55,000 RPM and can generate a maximum relative centrifugal force (RCF) of 300,000 x g. The MLA-55 rotor is made of a specialized alloy material to withstand the high rotational forces.
Automatically generated - may contain errors
Lab products found in correlation
Optima max xp ultracentrifuge, by Beckman Coulter (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Ultracentrifuge tube, by Beckman Coulter (1 mentions)
Optima max up, by Beckman Coulter (1 mentions)
Polypropylene tubes, by Beckman Coulter (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Merck Group (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Optima max xp, by Beckman Coulter (1 mentions)
Hd 2200, by Bandelin (1 mentions)
7 protocols using mla 55 rotor
1
Exosome Isolation and Purification
2
Preparation of Liposomal Formulations
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SUV was prepared as previously described28 (link),85 (link) with slight modifications of the protocol. Briefly, a dried film of egg PC (Kewpie, Tokyo, Japan) with or without 1-palmitoyl-2-oleoyl PS (NOF, Tokyo, Japan) or cholesterol (Sigma-Aldrich) was hydrated in 10 mM Tris-HCl buffer (150 mM NaCl, 0.02% NaN3, pH 7.4), followed by sonication on ice under nitrogen. To separate any remaining large vesicles, the resulting samples were centrifuged in a Beckman MLA-55 rotor for 1.5 h at 15 °C at 40,000 rpm.
3
Plasma Extracellular Vesicle Isolation
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All ultracentrifugations were performed using a Beckman Coulter Optima MAX-UP ultracentrifuge (stopping without break), with centrifugation durations based on a “50 nm cut-off size”, as described in Livshits et al., with an additional 5 min added to allow the rotor to come up to speed. Plasma samples were defrosted and transferred to a 13.5 ml Beckman Coulter ultracentrifuge tube (Prod. No. 355630) [30 (link)]. The plasma was diluted to 8.6 ml with particle-free PBS for ultracentrifugation. The tubes were centrifuged at 120000 g (RCF avg, 40800 rpm) for 2 hours at 20°C, using a Beckman Coulter MLA-55 rotor. The supernatant was removed, and the EV pellet was resuspended in 100 μl residual PBS.
4
Enrichment of small extracellular vesicles
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We used the established sUC method for enrichment of small EVs [61 (link)]. In short, sera aliquots were first defrosted on ice and centrifuged at 10,000× g for 20 min at 4 °C to remove any large extracellular particles. Next, 2 mL of 20% sucrose (sucrose (Merck Millipore, Burlington, MA, USA) in Dulbecco’s phosphate-buffered saline (dPBS, Sigma-Aldrich, St. Louis, MO, USA) was pipetted in polypropylene tubes (Beckman Coulter, Brea, CA, USA) and overlaid with diluted serum (1 mL of serum, mixed with 8.5 mL of particle-free dPBS). Samples were ultracentrifuged at 100,000× g for 135 min at 4 °C (MLA-55 rotor, Beckman Coulter, USA) and supernatant was aspirated from the tubes and walls of the tubes dried by low-lint highly absorbent paper. Finally, the pellet containing isolated EVs was fully resuspended in 200 µL of dPBS, mixed with 800 µL Tri-reagent (Sigma-Aldrich, USA), and stored at −20 °C.
5
Isolation and Characterization of Scc2-Scc4 Cohesin Complex
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S. pombe Scc2–Scc4 (200 nM) and cohesin (80 nM) were mixed and incubated at 4 °C for 30 min in 120 μl of buffer GG (20 mM Hepes-KOH (pH 7.5), 0.5 mM TCEP, 25 mM NaCl). A measure of 100 μl of the mixture was mounted on 5 ml of 20–50% glycerol gradient in buffer GG. The protein complexes were separated by ultracentrifugation at 4 °C at 48,000 r.p.m. for 15 h (beckman, MLA-55 rotor). The samples were fractionated by 200 μl each and analysed by SDS–PAGE and silver staining. Grafix were performed as the same protocol above but containing a linear gradient of 0–0.1% glutaraldehyde. Equivalent fractions from native and Grafix conditions were used for EM analyses.
6
Scc2-Scc4 Cohesin Complex Purification
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WT and mutant S. cerevisiae Scc2–Scc4 or GD0 domain (400 nM) were mixed and incubated with cohesin (100 nM) at 4 °C for 30 min in 120 μl of buffer GG (20 mM Hepes-KOH (pH 7.5), 0.5 mM TCEP, 25 mM NaCl). A measure of 100 μl of the mixture was mounted on 5 ml of 20–50% glycerol gradient in buffer GG. The protein complexes were separated by ultracentrifugation at 4 °C at 44,000 r.p.m. for 18 h (beckman, MLA-55 rotor). The samples were fractionated by 400 μl each and analysed by SDS–PAGE and silver staining.
7
Plasma Membrane Protein Extraction from Cultured Neurons
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Plasma membrane protein extraction was carried out as previously described.54 (link) Briefly, the cultured cortical neurons were washed 3 times with ice-cold PBS, and the neurons were harvested with a cell scraper. After centrifugation at 500g for 5 min at 4°C, the pellet was resuspended in ice-cold homogenization buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris-HCl buffer, pH 7.2, containing 1:100 diluted protease and phosphatase inhibitor cocktail, Sigma Aldrich). The neurons were then sonicated 15 s using a probe sonicator (Bandelin SONOPULS HD 2200), centrifuged at 10 000g for 10 min at 4°C, and the supernatant was collected. The sonication and supernatant extraction was done twice. The supernatant was centrifuged at 25 000g in an Optima MAX-XP benchtop ultracentrifuge with an MLA-55 rotor (Beckman Coulter Inc., Brea, CA) for 1 h at 4°C and the pellet was collected and resuspended in starting buffer (225 mM mannitol, 75 mM sucrose, and 30 mM Tris-HCl, pH 7.4). The suspension was centrifuged for 20 min at 25 000g, and the pellet was collected and lysed with 1× sample loading buffer (0.3 M sucrose, 2% SDS, 2.5 mM EDTA, 60 mM Tris, pH 8.8, 0.05% (w/v) bromophenol blue, 25 mM DTT), followed by heating at 95°C for 10 min.
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