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. 2021 Nov 18;22(22):12436. doi: 10.3390/ijms222212436

Figure 3.

Figure 3

Experimental design of the method comparison using five different methods for uEV preparation and a sixth method for one of the void urine samples. According to published protocols, void urine samples were differently pre-processed, either at 10,000× g or at 17,000× g. All details for the next procedures are provided. Following uEV preparation, uEV samples were analyzed by IFCM, NTA, Western blot, and transmission electron microscopy. Methods were: PEG precipitation followed by ultracentrifugation (PEG-UC) [31], PEG precipitation followed by size exclusion chromatography (PEG-SEC) [19], ultracentrifugation followed by size exclusion chromatography (UC-SEC) [15], ultrafiltration followed by size exclusion chromatography UF-SEC [32], and the commercial ExoEasy Maxi Kit (Qiagen), which is based on membrane affinity (ExoEasy). The sixth method was ultrafiltration followed by immunoaffinity capturing with commercial anti-CD9, anti-CD63, and anti-CD81 antibody-conjugated magnetic beads (UF-MACS [33]; it was only used for the preparation of EVs from one of the void urine samples.