Journal: Scientific reports

Article Title: Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

doi: 10.1038/s41598-024-62783-9

Figure Lengend Snippet: Figure 2. Characterization of small EVs (SEVs) isolated from 25 min (UC25min), 48 min (UC48min) and 60 min (UC60min) ultracentrifugation. (a) Nanoparticle tracking analysis (NTA) graphs display in y-axis: concentration (Particle/mL), and in x-axis: size (nm). (b) Western-blot (WB) for CD9, CD81, CD63, Flotillin-1, Alix and THP on SEVs samples. Western-blot images were cropped; the original blots are presented in Fig. S7 from Supplementary Material S2. (c) Transmission electron microscopy (TEM) analysis, in which black and red arrows correspond to SEVs and THP polymer (defined as the typical structure usually observed for THP protein), respectively. White bar represents a 200 nm scale. NTA and WB techniques were performed in 3 urine samples (only one sample is represented), and TEM in one sample.

Article Snippet: Subsequently, membranes were cut (before hybridization with antibodies) and incubated during 1h, at RT, with primary antibodies against CD9 (1:500; sc-13118, Santa Cruz Biotechnology, Texas, USA), CD81 (1:500; sc-166029, Santa Cruz Biotechnology, Texas, USA), CD63 (1:300; sc-5275, Santa Cruz Biotechnology), Flotillin-1 (1:300 sc-74566, Santa Cruz Biotechnology, Texas, USA), Alix (1:300; sc-53540, Santa Cruz Biotechnology, Texas, USA), THP (1:500; sc-271022, Santa Cruz Biotechnology, Texas, USA), Cytochrome C (1:500, sc-13560, Santa Cruz Biotechnology, Texas, USA) and Lamin A/C (1:500, sc-376248, Santa Cruz Biotechnology, Texas, USA) followed by incubation with a secondary antibody (1:5000; 7076S, Cell Signaling Technology, MA, USA) during 1h, at RT.

Techniques: Isolation, Concentration Assay, Western Blot, Transmission Assay, Electron Microscopy, Polymer

Journal: Scientific reports

Article Title: Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

doi: 10.1038/s41598-024-62783-9

Figure Lengend Snippet: Figure 3. Characterization from small (SEVs) and large EVs (LEVs) pellet comparing LEVs (LEVs) pelleting (UCLEVs) with urine supernatant filtering (UC48min). (a) Nanoparticle tracking analysis (NTA) graphics, in which y axis represents particle concentration (Particle/mL) and x-axis the size (nm). (b) Western-blot (WB) with antibodies against CD9, CD81, CD63, Flotillin-1, Alix and THP. Western-blot images were cropped; the original blots are presented in Fig. S8 from Supplementary Material S2. (c) Transmission electron microscopy (TEM) images from EVs samples, in which black and purple arrows indicates SEVs and LEVs respectively. White bar corresponds to a 200 nm scale. NTA and WB techniques were performed in 3 urine samples (only one sample is represented), and TEM in one sample.

Article Snippet: Subsequently, membranes were cut (before hybridization with antibodies) and incubated during 1h, at RT, with primary antibodies against CD9 (1:500; sc-13118, Santa Cruz Biotechnology, Texas, USA), CD81 (1:500; sc-166029, Santa Cruz Biotechnology, Texas, USA), CD63 (1:300; sc-5275, Santa Cruz Biotechnology), Flotillin-1 (1:300 sc-74566, Santa Cruz Biotechnology, Texas, USA), Alix (1:300; sc-53540, Santa Cruz Biotechnology, Texas, USA), THP (1:500; sc-271022, Santa Cruz Biotechnology, Texas, USA), Cytochrome C (1:500, sc-13560, Santa Cruz Biotechnology, Texas, USA) and Lamin A/C (1:500, sc-376248, Santa Cruz Biotechnology, Texas, USA) followed by incubation with a secondary antibody (1:5000; 7076S, Cell Signaling Technology, MA, USA) during 1h, at RT.

Techniques: Concentration Assay, Western Blot, Transmission Assay, Electron Microscopy

Journal: Scientific reports

Article Title: Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

doi: 10.1038/s41598-024-62783-9

Figure Lengend Snippet: Figure 4. Characterization of small EVs (SEVs) from protocols without (UC48min) and with washing step (UCwash). (a) Nanoparticle tracking analysis (NTA) graphic where x-axis represents particle size distribution (nm) and y-axis concentration (Particle/mL). (b) Western-blot (WB) for common EV markers including CD9, CD81, CD63, Flotillin-1 and Alix and for THP contaminant. Western-blot images were cropped; the original blots are presented in Fig. S7 from Supplementary Material S2. (c) Transmission electron microscopy (TEM) images from SEVs, in which black, blue, and orange arrows point out SEVs, THP-like polymer and protein precipitate, respectively. White bar indicates a 200 nm scale. NTA and WB techniques were performed in 3 urine samples (only one sample is represented), and TEM in one sample.

Article Snippet: Subsequently, membranes were cut (before hybridization with antibodies) and incubated during 1h, at RT, with primary antibodies against CD9 (1:500; sc-13118, Santa Cruz Biotechnology, Texas, USA), CD81 (1:500; sc-166029, Santa Cruz Biotechnology, Texas, USA), CD63 (1:300; sc-5275, Santa Cruz Biotechnology), Flotillin-1 (1:300 sc-74566, Santa Cruz Biotechnology, Texas, USA), Alix (1:300; sc-53540, Santa Cruz Biotechnology, Texas, USA), THP (1:500; sc-271022, Santa Cruz Biotechnology, Texas, USA), Cytochrome C (1:500, sc-13560, Santa Cruz Biotechnology, Texas, USA) and Lamin A/C (1:500, sc-376248, Santa Cruz Biotechnology, Texas, USA) followed by incubation with a secondary antibody (1:5000; 7076S, Cell Signaling Technology, MA, USA) during 1h, at RT.

Techniques: Concentration Assay, Western Blot, Transmission Assay, Electron Microscopy, Polymer

Journal: Scientific reports

Article Title: Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

doi: 10.1038/s41598-024-62783-9

Figure Lengend Snippet: Figure 5. Comparison between optimized differential ultracentrifugation (UC48min), density ultracentrifugation (dUC) and Exoquick (EXO) separation methods. (a) Nanoparticle tracking analysis (NTA) graphic represents particle size distribution in x-axis (nm) and concentration in y-axis (particle/mL). (b) Western-blot (WB) signals obtained from CD9, CD81, CD63, Flotillin-1, Alix and THP markers. Western- blot images were cropped; the original blots are presented in Fig. S9 from Supplementary Material S2. (c) Transmission electron microscopy (TEM) micrographs, in which the scale bar corresponds to 200 nm. Black and red arrows indicate small EVs (SEVs) and EXO polymer, respectively. NTA and WB techniques were performed in 3 urine samples (only one sample is represented), and TEM in one sample.

Article Snippet: Subsequently, membranes were cut (before hybridization with antibodies) and incubated during 1h, at RT, with primary antibodies against CD9 (1:500; sc-13118, Santa Cruz Biotechnology, Texas, USA), CD81 (1:500; sc-166029, Santa Cruz Biotechnology, Texas, USA), CD63 (1:300; sc-5275, Santa Cruz Biotechnology), Flotillin-1 (1:300 sc-74566, Santa Cruz Biotechnology, Texas, USA), Alix (1:300; sc-53540, Santa Cruz Biotechnology, Texas, USA), THP (1:500; sc-271022, Santa Cruz Biotechnology, Texas, USA), Cytochrome C (1:500, sc-13560, Santa Cruz Biotechnology, Texas, USA) and Lamin A/C (1:500, sc-376248, Santa Cruz Biotechnology, Texas, USA) followed by incubation with a secondary antibody (1:5000; 7076S, Cell Signaling Technology, MA, USA) during 1h, at RT.

Techniques: Comparison, Concentration Assay, Western Blot, Transmission Assay, Electron Microscopy, Polymer

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Extracellular Vesicle Marker Changes Associated With Disease Activity in Relapsing-Remitting Multiple Sclerosis

doi: 10.1212/NXI.0000000000200404

Figure Lengend Snippet: EVs were isolated by size-exclusion chromatography (SEC, SmartSEC, System Biosciences). (A) Analysis of the classical EV marker Alix by Western blotting after isolation. Equal amounts of EVs and plasma samples (10 µg) were used. A weak level of albumin was detected in the EV fraction. (B) Freshly isolated EVs were subjected to cryo-electron microscopy (cryo-EM). Cryo-electron microscopy observations showed EVs with typical lipid bilayer membrane. EV = extracellular vesicle.

Article Snippet: Thereafter, membranes were incubated with a primary antibody against Alix (1:500, NBP1-90201, Novus Biologicals, Centennial, CO) in a rolling shaker overnight at 4°C.

Techniques: Isolation, Size-exclusion Chromatography, Marker, Western Blot, Clinical Proteomics, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Membrane

Journal: iScience

Article Title: An antibiotic derivative as a new potential tool in the prevention of hemolytic uremic syndrome

doi: 10.1016/j.isci.2025.113076

Figure Lengend Snippet: Quantitative capillary western blot analysis of the proteins derived from human EVs Human EVs were isolated after treatment of human blood with vehicle or with 2 nM Stx2a in the absence or in the presence of 0.01 μg/mL NAB815 and their proteins extracted as described in . (A) Representative WES of the analyzed antigens (Alix, CD45, and CD42a) and associated proteins (Stx2a) is shown. (B–E) Data obtained with different human donors ( n = 4) represent the percentage (mean ± SD) of the quantitative determinations of the different antigens with respect to controls (C–E) or Stx2a (B). (F–H) Data obtained with different human donors ( n = 4) are expressed as percentage (mean ± SD) of the stimulation in the presence of Stx2a that was set at 100%. Different primary antibodies were used: rabbit polyclonal anti-Alix 1:50 (Novus Biological) as an EV marker, mouse monoclonal anti-CD45 1:250 (BD Transduction Laboratories) as a leukocyte marker, rabbit polyclonal anti-CD42a 1:10 (GeneTex) as a platelet marker, and rabbit polyclonal anti-Stx2a 1:50 (Dr. Stefano Morabito, ISS Rome) to detect the toxin. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (two-tailed unpaired t test). See also .

Article Snippet: Different primary antibodies were used: rabbit polyclonal anti-Alix 1:50 (Novus Biological) as an EV marker, mouse monoclonal anti-CD45 1:250 (BD Transduction Laboratories) as a leukocyte marker, rabbit polyclonal anti-CD42a 1:10 (GeneTex) as a platelet marker, and rabbit polyclonal anti-Stx2a 1:50 (Dr. Stefano Morabito, ISS Rome) to detect the toxin.

Techniques: Western Blot, Derivative Assay, Isolation, Marker, Transduction, Two Tailed Test