600 μg to 1 mg of total proteins from freshly lysed LCLs was loaded onto 10–50% sucrose gradients as previously described (45 (link)). The tubes were centrifuged at 4°C and at 36 000 rpm for 2 h in a SW41 rotor (Optima L100XP ultracentrifuge; Beckman Coulter). The gradient fractions were measured at OD254nm using a syringe pump and UV detector (Brandel) and collected with a Foxy Jr gradient collector (Teledyne Isco).
Optima l 100
Manufactured by Beckman Coulter
Sourced in United States
The Optima L-100 is a high-performance floor-standing ultracentrifuge designed for a wide range of laboratory applications. It features a compact footprint and advanced technology to provide efficient and reliable sample processing capabilities.
Automatically generated - may contain errors
4 protocols using optima l 100
1
Sucrose Gradient Fractionation of LCL Proteins
2
Polysome Profiling and RNA Analysis
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About 2 × 107cells were prepared to incubate with 100 μg/ml cycloheximide for 15 min, and then total lysates were layered onto 30% sucrose in ice-cold buffer containing 20 mm HEPES, pH 7.4, 50 mm potassium acetate, 5 mm magnesium acetate, 1 mm DTT, 1 unit of RNasin/μl, 1 μg of leupeptin/ml, 1 μg of aprotinin/ml, and 0.5 mm phenylmethylsulfonyl fluoride. The lysates were centrifuged at 30,000 rpm for 2 h at 4 °C (Beckman Optima L-100), and RNA was isolated from the pellet (polysomal fraction) and analyzed by RT-qPCR.
3
Isolation and Characterization of Exosomes from ESCs
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The culture supernatants of ESCs were harvested for exosome isolation by sequential centrifugation (Optima L-100 ultracentrifuge; Beckman Coulter, USA). Briefly, supernatants were centrifuged at 300 × g for 10 min followed by 2000 × g for 10 min at room temperature. Then, the supernatants were centrifuged at 10,000 × g for 30 min at room temperature to remove cell debris, followed by ultracentrifugation twice at 100,000 × g for 70 min at room temperature. The final pellets were exosomes. Transmission electron microscopy (TEM) using a JEOL (Tokyo, Japan) system was employed to identify exosome morphology. The size and concentration of exosomes were analyzed by Nanoparticle-tracking analysis (NTA) using an analyzer (NanoSight NS300; Malvern Instruments, Malvern, UK). Specific markers for exosomal proteins, CD9 and CD63, were identified by Western blotting.
4
Isolation and Purification of Exosomes
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One million CAFs were cultured on a 10-cm dish for 24 h with IMDM supplemented with 10% fetal bovine serum (FBS). Subsequently, cells were washed with phosphate-buffered saline (PBS) and then cultured with IMDM supplemented with 10% exosome-free FBS from which exosomes had been removed by ultracentrifugation at 32,000 rpm for 16 h (ultracentrifuge: Optima LE-80 or Optima L-100, Beckman Coulter, CA, USA; ultracentrifuge rotor: SW32Ti-12U, Beckman Coulter). After incubation for 48 h, conditioned medium (CM) was collected and centrifuged at 300 × g for 10 min at 4 °C. Then the supernatant was centrifuged at 2000 × g for 10 min at 4 °C. To thoroughly remove cellular debris, the supernatant was passed through a 0.22-μm filter. The CM was ultracentrifuged at 35,000 rpm using a SW41Ti-14E2457 rotor for 70 min at 4 °C or at 32,000 rpm using a SW32Ti-12U rotor for 84 min at 4 °C. The pellets containing exosomes were washed with 0.22-μm membrane-filtered PBS. PBS containing exosomes was ultracentrifuged at 35,000 rpm using the SW41Ti-14E2457 rotor for 70 min at 4 °C or at 32,000 rpm using a SW32Ti-12U rotor for 84 min at 4 °C. Finally, the pellets containing exosomes were resuspended in 0.22-μm membrane-filtered PBS.
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