IPNA clinical practice recommendations for the diagnosis and management of children with steroid-resistant nephrotic syndrome. were collected to new tubes, snap frozen in liquid nitrogen, and kept at ?80C for further use. At the time of ultracentrifugation, frozen tubes were thawed very slowly overnight at +4C to prevent EV and/or exosome rupture before isolation and vigorously 2,4-Pyridinedicarboxylic Acid vortexed before proceeding to ultracentrifugation. If needed, to adjust the volume to a fixed amount before centrifugation, some plasma samples were supplemented with cold PBS to 14 mL and centrifuged at 10,000 for 30 min at 4C. Supernatants were transferred to another ultracentrifuge tube and centrifuged at 100,000 for 90 min at 4C. At the end of this step, EVs were sedimented. The supernatants were accepted as plasma-depleted EVs and used in further experiments. To get rid of contaminating proteins, EVs were further washed with 14 mL of PBS and recentrifuged at 100,000 for 90 min at 4C. The pellets made up of EVs were dissolved with PBS. Determination of EV Protein Content and Number Protein content of EVs was decided with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturers instructions. Briefly, 25 L/well BSA standard dilutions and 5 L/well samples were placed in 96-well plates and mixed with 200 L of the working reagent. The plate was incubated in the dark at 37C for 30 min and then cooled down at room heat for 15 min. Absorbance at 562 nm was measured with a Synergy HT microplate reader (BioTek, Winooski, VT). The particle number of 2,4-Pyridinedicarboxylic Acid 1 1 g/L EVs was measured by a tunable pulse resistive index system (QNano, Izon Biosciences). Protein amount and particle concentration of EVs are presented as normalized to plasma volume (mL) and plasma albumin concentration (g/dL). Transmission Electron Microscopy The morphology and size of EVs were evaluated by transmission electron microscopy (TEM). EV suspensions (5 L) were decreased onto formvar/carbon-coated nickel mesh grids and incubated for 20 min. Excess suspension around the nickel mesh grids was then blotted with filter paper, and nickel mesh grids were negatively stained with 2.0% phosphotungstic acid and 2.0% uranyl acetate, respectively. After being washed, samples were air-dried for 15 min and FGFR2 visualized using a digital camera (Orius) connected to transmission electron microscope (JEM1400, Jeol, Tokyo, Japan). Analysis of EV Surface Markers by Flow Cytometry A bead-based detection method was used for surface protein characterization of EVs with flow cytometry. Latex beads were coated with purified anti-human CD63 or CD81 antibody to capture EVs. Carboxyl latex beads (Thermo Fisher Scientific) were mixed with anti-CD63 (clone H5C6, BioLegend, San Diego, CA) or anti-CD81 (clone 5A6, BioLegend) at a 1 L:1 g (bead-to-antibody) ratio to capture CD63- or CD81-positive EVs. The volume was completed to 50 L with PBS, and the mixture was incubated for 30 min at room temperature. The volume was then increased to 500 L with PBS, and the mixture was incubated on a rotator at a low speed overnight. The bead-antibody mixture was precipitated at 10,000 for 10 min, blocked with 5% BSA (Capricorn Scientific, Ebsdorfergrund, Germany) for 4 h at room temperature, precipitated again at 10,000 for 10 min, resuspended in PBS made up of 1% BSA, and stored at 4C. For each staining of EVs, 1 g of EVs was 2,4-Pyridinedicarboxylic Acid mixed with 1 L of the final coated bead answer. The volume was increased with PBS up to 50 L, and the mixture was incubated at room 2,4-Pyridinedicarboxylic Acid temperature for 30 min. The volume was then increased to 500 L, and the mixture was slowly rotated overnight to let the EVs and beads conjugate. After overnight conjugation, EV-bead conjugates were incubated with fluorochrome antibodies for human CD9 (clone HI9a, Biolegend), CD63, CD81 (Biolegend), and PE-podocalyxin (clone 3D3, Santa Cruz Biotechnology, Dallas, TX) at 1:50 dilution and their appropriate isotype controls at a concentration 2,4-Pyridinedicarboxylic Acid of 1 1 g/mL in 100 L volume for 1 h at room temperature in the dark. After 1.