Hydrostatic Filtration Dialysis (HFD): A schematic representation of the methodology used for vesicle isolation is shown in figure 1 . Pooled urine samples (50 ml per tube) were centrifuged at a Relative Centrifugal Force (RCF) of 2,000 g calculated at average radius of 100 mm in a swing bucket rotor Benchtop Universal 320 centrifuge (Hettich Zentrifugen, Tuttingen, Germany) for 30 min at room temperature (RT) (without braking). The supernatant (SN) ~ 0.5l was poured in a separating funnel connected with a dialysis membrane made of cellulose ester (CE) with molecular weight cut-off (MWCO) of 1,000 kDa (Spectra/Por Biotech MWCO 1,000,000 MWCO Catalogue number 131486; Spectrum Laboratories, Ca) (Supplemental Fig. 1 ).
The hydrostatic pressure of the urinary solution in the funnel pushes the solvent (water) through the mesh of dialysis membrane (filtration), together with all the analytes below the selected MWCO. After the first step resulting in sample concentration, the separating funnel was refilled with 200 ml of deionised filtrate (0.22 µm) water (R ≥ 18.2 MΩ·cm, mQ water) to rinse away remaining analytes below the MWCO until the volume of 5–8 ml of volume is reached. This filtration-concentration-dialysis process is called “hydrostatic filtration dialysis” (HFD).
HFD and differential centrifugation:The retained solution above the 1,000 kDa cut-off (HFDa) (5 ml) was then centrifuged at 5,000 g, 20,000 g and/or 40,000 g calculated at maximum radius 105 mm of a fixed angle JA-20 rotor (clearing factor or k factor = 770) (Beckman Coulter, Fullerton, Ca) for 1 h at RT. The retained 40,000 g supernatant (SN) fraction (5 ml) was then ultracentrifuged at 200,000 g calculated at maximum radius 91.9 mm of 70 Ti fixed-angle rotor (k factor = 44) (Beckman Coulter) for 2 h (RT) using a Beckman XL-80 Ultracentrifuge (Beckman Coulter). All the pellets were re-suspended in mQ water. For a pilot study, HFDa from the starting urinary volumes of 15, 50, 100 and 200 ml were concentrated to 3 ml. After determining the protein concentration, an equal amount of total protein was loaded in polycarbonate centrifugation tubes (3 ml). Ultracentrifugation was performed at 200,000 g calculated at the maximum radius 82.0 mm of 70.1Ti fixed-angle rotor (k factor = 36) (Beckman Coulter) for 2 h at RT.
Differential centrifugation and HFD: Comparative analysis was performed according to Fernández-Llama and colleagues15 (link). Pellets from 2,000 g and 17,000 g were resuspended in 10 ml of 250 mM sucrose, 10 mM triethanolamine pH7.6 and 200 mg/ml of DTT for 10 minutes at 37°C vortexing every 2 minutes. Centrifugations at 17,000 g (42 ml per tube of urine and 10 ml of dithiothreitol (DTT) fraction) were performed in a fixed angle JA-20 rotor (clearing factor or k factor = 770) (Beckman Coulter) for 30 min at RT. RCF were calculated at average radius of 70 mm. Ultracentrifugations (16,5 ml urine per tube and 10 ml of DTT fraction) were performed at 200,000 g calculated at maximum radius 91.9 mm of 70 Ti fixed-angle rotor (k factor = 44) (Beckman Coulter) for 2 h (RT) using a Beckman XL-80 Ultracentrifuge (Beckman Coulter). All the final pellets were resuspended in 1 ml of purified water. The final SNs were poured in HFD system and processed as described above. Conversely, the urine solution below the 1,000 kDa cut-off (HFDb) was ultracentrifuged (16.5 ml per tube) at 200,000 g calculated at maximum radius 91.9 mm of 70 Ti fixed-angle rotor (k factor = 44) (Beckman Coulter) for 2 h (RT) using a Beckman XL-80 Ultracentrifuge (Beckman Coulter).
The hydrostatic pressure of the urinary solution in the funnel pushes the solvent (water) through the mesh of dialysis membrane (filtration), together with all the analytes below the selected MWCO. After the first step resulting in sample concentration, the separating funnel was refilled with 200 ml of deionised filtrate (0.22 µm) water (R ≥ 18.2 MΩ·cm, mQ water) to rinse away remaining analytes below the MWCO until the volume of 5–8 ml of volume is reached. This filtration-concentration-dialysis process is called “hydrostatic filtration dialysis” (HFD).
HFD and differential centrifugation:The retained solution above the 1,000 kDa cut-off (HFDa) (5 ml) was then centrifuged at 5,000 g, 20,000 g and/or 40,000 g calculated at maximum radius 105 mm of a fixed angle JA-20 rotor (clearing factor or k factor = 770) (Beckman Coulter, Fullerton, Ca) for 1 h at RT. The retained 40,000 g supernatant (SN) fraction (5 ml) was then ultracentrifuged at 200,000 g calculated at maximum radius 91.9 mm of 70 Ti fixed-angle rotor (k factor = 44) (Beckman Coulter) for 2 h (RT) using a Beckman XL-80 Ultracentrifuge (Beckman Coulter). All the pellets were re-suspended in mQ water. For a pilot study, HFDa from the starting urinary volumes of 15, 50, 100 and 200 ml were concentrated to 3 ml. After determining the protein concentration, an equal amount of total protein was loaded in polycarbonate centrifugation tubes (3 ml). Ultracentrifugation was performed at 200,000 g calculated at the maximum radius 82.0 mm of 70.1Ti fixed-angle rotor (k factor = 36) (Beckman Coulter) for 2 h at RT.
Differential centrifugation and HFD: Comparative analysis was performed according to Fernández-Llama and colleagues15 (link). Pellets from 2,000 g and 17,000 g were resuspended in 10 ml of 250 mM sucrose, 10 mM triethanolamine pH7.6 and 200 mg/ml of DTT for 10 minutes at 37°C vortexing every 2 minutes. Centrifugations at 17,000 g (42 ml per tube of urine and 10 ml of dithiothreitol (DTT) fraction) were performed in a fixed angle JA-20 rotor (clearing factor or k factor = 770) (Beckman Coulter) for 30 min at RT. RCF were calculated at average radius of 70 mm. Ultracentrifugations (16,5 ml urine per tube and 10 ml of DTT fraction) were performed at 200,000 g calculated at maximum radius 91.9 mm of 70 Ti fixed-angle rotor (k factor = 44) (Beckman Coulter) for 2 h (RT) using a Beckman XL-80 Ultracentrifuge (Beckman Coulter). All the final pellets were resuspended in 1 ml of purified water. The final SNs were poured in HFD system and processed as described above. Conversely, the urine solution below the 1,000 kDa cut-off (HFDb) was ultracentrifuged (16.5 ml per tube) at 200,000 g calculated at maximum radius 91.9 mm of 70 Ti fixed-angle rotor (k factor = 44) (Beckman Coulter) for 2 h (RT) using a Beckman XL-80 Ultracentrifuge (Beckman Coulter).
