0.1 m filters

For isolation of urinary m/lEVs, we modified a urinary exosome extraction protocol (Fernandez-Llama et al., 2010) (link). The centrifugation conditions were identical for plasma and urine so that the size and the density of m/lEVs were similar, enabling comparison of plasma and urinary m/lEVs.
In flow cytometric analysis, the volume of urine used for each assay was 1.2 mL from each donor. In electron microscopy, the volume of urine used was 15 mL. Samples were independent and were treated individually prior to each measurement. Collected urine was centrifuged at 2,330×g for 10 min twice. The supernatant was centrifuged at 18,900×g for 30 min in a fixed-angle rotor. The m/lEV pellet obtained from centrifugation was reconstituted by vortex mixing (1-2 min) with 0.2 mL of DPBS followed by incubation with DTT (final concentration 10 mg/mL) at 37 • C for 10-15 min. The samples were centrifuged again at 18,900×g for 30 min and the supernatant was discarded. Addition of DTT, a reducing agent, reduced the formation of Tamm-Horsfall protein (THP) polymers. THP monomers were removed from m/lEVs after centrifugation. DTT-containing DPBS solutions were filtered through 0.1-µm filters (Millipore).

After resuspending m/lEV pellets in 60 µL of DPBS, we added saturating concentrations of several labelled antibodies, Annexin V and normal mouse IgG and incubated the tubes in the dark, without stirring, for 15-30 min at room temperature. In one case, we added labelled antibodies directly to 60 µL of PFP for staining. We resuspended stained fractions in Annexin V binding buffer (BD Biosciences: 10 mM HEPES, 0.14 mM NaCl, 2.5 mM CaCl 2 , pH 7.4) for analysis by flow cytometry. DPBS and Annexin V binding buffer were filtered through 0.1-µm filters (Millipore). Flow cytometry was performed using a FACSVerse TM flow cytometer (BD Biosciences). The flow cytometer was equipped with 405 nm, 488 nm and 638 nm lasers to detect up to 13 fluorescent parameters. The flow rate was 12 µL /min. Forward scatter voltage was set to 381, side scatter voltage was set to 340, and each threshold was set to 200. Details of excitation (Ex.) and emission (Em.) wavelengths as well as voltages described in supplements Fig. Flow cytometry was performed using FACSuite TM software (BD Biosciences) and data were analyzed using FlowJo software. The authors have applied for the following patents for the characterization method of m/lEVs isolated from plasma and urine with a flow cytometer: JP2018-109402(plasma) and JP2018-109403(urine).