Urine samples were pre-processed, as indicated above. Then, 200 µL of PBS (ThermoFisher Scientific/Gibco, Carlsbad, CA, USA) were added to 7.8 mL pre-processed urine. Following addition of 2 mL 50% w/v PEG-6000 solution (Sigma-Aldrich; final concentration: 20%), uEVs were precipitated at 4 °C overnight. Precipitates were pelleted by centrifugation for 35 min at 1500× g and 4 °C. Next, size exclusion chromatography was performed with 10 mL of Sepharose CL-2B (GE Healthcare, Uppsala, Sweden) using self-packed, PBS-equilibrated columns (Econo-Pac 20 mL, Bio-Rad Laboratories, Hercules, CA, USA), according to the “Mini-SEC” procedure reported by Hong and colleagues [50 (link)]. The resuspended uEV pellet was applied onto the top mesh of the size exclusion columns. Six fractions, each of 1 mL, were eluted. After elution of each fraction, 1 mL PBS was added to the column. Fraction 4, the fraction containing most EVs, was used for all downstream analyses.
Peg 6000 solution
Manufactured by Merck Group
PEG-6000 solution is a laboratory reagent used in various biological and chemical applications. It is a polyethylene glycol solution with a molecular weight of 6,000 Daltons. The solution serves as a functional component in experimental procedures, providing specific physicochemical properties that may be required for the research or analysis being conducted. The detailed description and intended uses of PEG-6000 solution should be provided by subject matter experts to ensure an accurate and unbiased representation.
Automatically generated - may contain errors
2 protocols using peg 6000 solution
1
Urine-derived Extracellular Vesicle Isolation
2
Urine Extracellular Vesicle Isolation
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Exactly as before, urine samples were pre-processed. 200 µL of PBS (ThermoFisher Scientific / Gibco, Carlsbad, CA, USA) were added to 7.8 mL pre-processed urine. Following addition of 2 mL 50% w/v PEG-6000 solution (Sigma-Aldrich; final concentration: 20%) uEVs were precipitated at 4°C overnight. Precipitates were pelleted by centrifugation for 35 minutes at 1,500 x g and 4°C. Next, size exclusion chromatography was performed with 10 mL of Sepharose CL-2B (GE Healthcare, Uppsala, Sweden) using self-packed and PBS-equilibrated columns (Econo-Pac 20 mL, Bio-Rad Laboratories, Hercules, CA, USA) according to the "Mini-SEC" procedure reported by Hong and colleagues (31) . The resuspended uEV pellet was applied onto the top mesh of the size exclusion columns. 6 fractions, each of 1 mL, were eluted.
After elution of each fraction, 1 mL PBS was added to the column. Fraction 4, the fraction containing most EVs, was used for all downstream analyses.
After elution of each fraction, 1 mL PBS was added to the column. Fraction 4, the fraction containing most EVs, was used for all downstream analyses.
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