Exosome Dashboard

OptiPrep™ (iodixanol)

The optimum density gradient medium for purifying exosomes

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OptiPrep™ is a sterile, endotoxin tested solution of 60% iodixanol in water with a density of 1.32 g/ml.

Iodixanol was developed as an X-ray contrast medium and has therefore been subjected to rigorous clinical testing. It is non-ionic, non-toxic to cells and metabolically inert. Iodixanol solutions can be made iso-osmotic at all useful densities. Iodixanol solutions have low viscosity and osmolality.

Optiprep™ for exosome purification using high-resolution density gradient ultracentrifugation

High-resolution (12%–36%) iodixanol density gradient fractionation separates membrane-enclosed sEVs from non-vesicular (NV) components

OptiPrep™ (iodixanol) density media were prepared in ice-cold PBS immediately before use to generate discontinuous step (12%–36%) gradients. Crude pellets of sEVs (P120) were resuspended in ice-cold PBS and mixed with ice-cold iodixanol/PBS for a final 36% iodixanol solution. The suspension was added to the bottom of a centrifugation tube and solutions of descending concentrations of iodixanol in PBS were carefully layered on top yielding the complete gradient. Identical gradients without sample were generated in the same manner for later determination of fraction densities. The bottom-loaded 12%–36% gradients were subjected to ultracentrifugation at 120,000 x g for 15h at 4°C using a SW41 TI Swinging Bucket rotor (k factor of 124, Beckman Coulter). Twelve individual fractions of 1 mL were collected from the top of the gradient. From the duplicate gradient, fraction densities were measured using a refractometer (ATAGO, Tokyo, Japan). For Nanoparticle Tracking Analysis (NTA), fractions were pooled and diluted in particle-free PBS. For immunoblotting, each individual 1 mL fraction was transferred to new ultracentrifugation tubes, diluted 12-fold in PBS and subjected to ultracentrifugation at 120,000 x g for 4h at 4°C using a SW41 TI swinging bucket rotor. The resulting pellets were lysed in cell lysis buffer for 30 min on ice. For RNA and DNA extraction, fractions were pooled, transferred to new ultracentrifugation tubes, diluted approximately 6-fold in PBS and subjected to ultracentrifugation at 120,000 x g for 4h at 4°C using a SW 32 Ti Swinging Bucket rotor.

(from: Jeppesen, D., Fenix, A., Franklin, J., Higginbotham, J., Zhang, Q., Zimmerman, L., Liebler, D., Ping, J., Liu, Q., Evans, R., Fissell, W., Patton, J., Rome, L., Burnette, D., Coffey, R. (2019). Reassessment of Exosome Composition Cell 177(2), 428-445.e18.)

Optiprep™ Application Notes

1. Mammalian cell exosomes and other microvesicles from conditioned medium
2. Extracellular vesicles from non-mammalian sources
3. Intracellular exocytic vesicle trafficking and exocyst complex – a short methodological summary
4. Preparation of synaptosomes, synaptoneurosomes, neuromelanin granules and synaptic vesicles – a methodological survey
5. Isolation of vesicular and granular fractions from various sources