Figure 3 Comparison of approaches to isolate extracellular vesicles

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Figure 3 Comparison of approaches to isolate extracellular vesicles from the urine of healthy individuals and from patients with nephrotic syndrome Figure 3 | Comparison of approaches to isolate extracellular vesicles from the urine of healthy individuals and from patients with nephrotic syndrome. Urine is collected as a spot or timed urine sample (part a). The process of urinary extracellular vesicle isolation begins with a low speed and/or low centrifugal force (3,000 g) centrifugation step for a short time (≤10 min) and at low temperature (4oC) to clarify the urine (that is, remove the flocculent material, which can include bacteria and cells). Ideally, the urine is carried forward (part b) through to the urinary extracellular vesicle isolation step. This step represents a dynamic field of investigation, and includes methods such as differential centrifugation or ultracentrifugation, single step centrifugation using density gradient material (sucrose, Percoll), filtration or ultrafiltration, precipitation (for example, Exoquick), immunoaffinity capture and hydrostatic dialysis. The complex composition of urine from patients with nephrotic syndrome interferes with the isolation of urinary extracellular vesicles, and additional steps (such as density gradient centrifugation or size exclusion chromatography (SEC)) are required (part c) to further purify the urinary extracellular vesicles from contaminating high-molecular-weight protein complexes (such as albumin) that co-isolate with the urinary extracellular vesicles. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; D2O, deuterium oxide; DTT, dithiothreitol; HPLC, high performance liquid chromatography; MWCO, molecular weight cut-off; Q, Qiagen; SB, Systems Biosciences; TFS, ThermoFisher Scientific; VVLP, hydrophilic polyvinylidene difluoride. Merchant, M. L. et al. (2017) Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery Nat. Rev. Nephrol. doi:10.1038/nrneph.2017.148