Journal: Transfusion Medicine and Hemotherapy

Article Title: Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation

doi: 10.1159/000508712

Figure Lengend Snippet: FACS-based characterization of isolated EVs (second round). A FACS histograms depicting the relative fluorescence/marker intensity of EV preparation 2.1 (black line) against unstained EV particle control (grey line). B Corresponding marker expression in HCT116 cells (extracellular staining for CD9, CD63, and CD81 and intracellular staining for Alix, TSG101, and calnexin). C, E Mean fluorescence intensity (MFI) raw values of CD63 (C) and CD81 (E) marker expression from laboratories 2.1–2.4. D, F MFI values per particle concentration of CD63 (D) and CD81 (F; left y axis) against the respective particle concentration per ml CCM (right y axis).

Article Snippet: HCT116-derived EVs were captured on anti-human CD9 beads for flow detection (Exosome-Human CD9 beads; Thermo Fisher Scientific).

Techniques: Isolation, Fluorescence, Marker, Control, Expressing, Staining, Concentration Assay

Journal: Physiological reports

Article Title: Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training.

doi: 10.14814/phy2.16016

Figure Lengend Snippet: FIGURE 3 EV surface marker expression is altered by AHRET in men and women. The proportion of EVs expressing SGCA (a), CD9 (b), VAMP3 (c), and THSD (d) were measured via Imaging Flow Cytometry and are shown as percentage of total gated EVs. Representative images of EV particles with bright-field (BF) image of each particle and subsequent fluorescent channel images showing presence or absence of muscle-derived EVs (SGCA+), microvesicles (VAMP3+), exosomes (CD9+) and apoptotic bodies (THSD+) vesicles are shown (e). N = 9. Statistical testing was done via three-way ANOVA.

Article Snippet: Samples were then stained with the following antibodies and dilutions: anti- human CD9 Alexa Fluor 700 (1:300 dilution; Novus Biologicals, CO), anti- human VAMP3 Alexa Fluor 405 (1:300; Novus Biologicals), anti- human thrombospondin (THSD- 1) Alexa Fluor 594 (1:100; Novus Biologicals), alpha sarcoglycan (SGCA) FITC (1:400; Biorbyt, St Louis, MO).

Techniques: Marker, Expressing, Imaging, Flow Cytometry, Derivative Assay

Journal: Non-Coding RNA

Article Title: Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein

doi: 10.3390/ncrna1020151

Figure Lengend Snippet: Enrichment of urinary miR-16, miR-192 and classical exosomal markers in the same sucrose gradient fractions. Following overnight sucrose gradient ultracentrifugation of the sedimented urinary pellet at 210,000 g at 4 °C, enrichment was observed in sucrose gradient fractions 3–5 of ( a ) multivesicular body markers TSG101 and Alix detected by Western analysis ( b ) exosome-expressed tetraspanins CD9 and CD81 detected by flow cytometry and ( c ) RT-qPCR-detected miR-16 and miR-192.

Article Snippet: Primary monoclonal antibodies (2–10 μg/mL) against CD9 (R&D Systems, Abingdon, Oxfordshire, UK) and CD81 (AbD Serotec, Kidlington, Oxfordshire, UK) were used for 1 h at room temperature.

Techniques: Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: Biology

Article Title: Characterization of Rabbit Mesenchymal Stem/Stromal Cells after Cryopreservation.

doi: 10.3390/biology12101312

Figure Lengend Snippet: Figure 5. Representative histograms of MSC surface marker expression of CD9 and CD44, and hematopoietic marker expression of CD34 and CD45. The red histogram represents isotype control, and the green histogram represents respective antibodies. MSCs, mesenchymal stem/stromal cells; ADP, adipose tissue; BM, bone marrow; BF, before cryopreservation; AF, after cryopreservation.

Article Snippet: CD9 (catalog no. NBP1-28364, Novus Biologicals, Centennial, CO, USA) and CD44 (catalog no. bs-0521R-FITC, Bioss, Woburn, MA, USA) were used as MSC surface marker antibodies, and CD34 (catalog no. bs-0646R-FITC, Bioss, Woburn, MA, USA) and Biology 2023, 12, 1312 5 of 23 CD45 (catalog no. MHCD450, Thermo Fisher Scientific Inc., Life Technologies Corporation, Frederick, MD, USA) were used as hematopoietic cell marker antibodies [40–44].

Techniques: Marker, Expressing, Control

Journal: Alzheimer's Research & Therapy

Article Title: Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

doi: 10.1186/s13195-022-01073-w

Figure Lengend Snippet: Immuno-digital ICA (idICA) for counting Aß-bound EVs. A , B Workflow of immune-digital ICA analysis of Aß-bound EVs. Ganglioside GM1-containing EVs are captured by cholera toxin B subunit (CTB)-coated magnetic beads (MB) and then reacted with the DNA oligo-conjugated detection antibody against an exosome marker protein CD9 or Aß ( A ). The resultant EV–bead–antibody complex in A and substrates for ICA are loaded into the digital device, enclosed into individual microwells by fluorinated oil, and analyzed by fluorescent imaging after the ICA reaction at 66 °C for 15 min ( B ). C Schematic illustration of ICA. (1) Invasive oligonucleotides and probe oligonucleotides hybridize to target DNA and generate 5′-flap structures in the probe oligonucleotides, which are cleaved by FEN-1. The target DNA is conjugated to detection antibodies. (2) The cleaved 5′-flaps bind to fluorescent probes and form 5′-flap structures between a quencher molecule [Q] and a fluorophore [F]. Cleavage of 5′-flaps by FEN-1 emits fluorescence signals. Unannealed 5′-flaps are shown in blue and yellow. Arrows indicate 5′-flap cleavage by FEN-1. D The digital device used in this study. There are 100 blocks of well arrays; 10,000 wells in each block correspond to the 10 6 microwells on a single fabricated device. Phase-contrast image and fluorescent image of a block of well array are shown. Scale bars, 1 mm

Article Snippet: Monoclonal antibodies against CD9 (MAB5218, R&D Systems, Minneapolis, MN), Aß (BAN50, FUJIFILM, Tokyo, Japan), and ßIII tubulin (#2146, Cell Signaling, Danvers, MA) were used.

Techniques: Magnetic Beads, Marker, Imaging, Fluorescence, Blocking Assay

Journal: Alzheimer's Research & Therapy

Article Title: Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

doi: 10.1186/s13195-022-01073-w

Figure Lengend Snippet: Quantification of GM1-containing EVs Using the idICA. A Western blot analysis of CD9, ganglioside GM1, and ßIII tubulin in N2a cell lysates (1 × 10 5 cells/lane) and EVs (1 × 10 7 cells/lane). B Representative fluorescent images of various concentrations of N2a-derived EVs in the idICA, which is constructed from CTB capture and anti-CD9 detection. Each image shows a block of well array corresponding to 10,000 microwells. Scale bar, 200 μm. C , D The ratio of fluorescent beads to trapped beads in a block of well array is plotted as the concentration of CD9 captured on CTB-coated beads (CTB-CD9) in N2a-derived EVs. Plots on the semi-logarithmic ( C ) and linear ( D ) scales are shown. Data represent mean ± SD ( n = 3 each)

Article Snippet: Monoclonal antibodies against CD9 (MAB5218, R&D Systems, Minneapolis, MN), Aß (BAN50, FUJIFILM, Tokyo, Japan), and ßIII tubulin (#2146, Cell Signaling, Danvers, MA) were used.

Techniques: Western Blot, Derivative Assay, Construct, Blocking Assay, Concentration Assay

Journal: Alzheimer's Research & Therapy

Article Title: Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

doi: 10.1186/s13195-022-01073-w

Figure Lengend Snippet: Quantification of Aß-bound and GM1-containing EVs using the idICA. A Western blot analysis of Aß, ganglioside GM1, and ßIII tubulin in APP-N2a cell lysates (1 × 10 5 cells/lane) and EVs (1 × 10 7 cells/lane). B Representative fluorescent images of various concentrations of APP-N2a-derived EVs in the idICA, which is constructed from CTB capture and anti-Aß detection. Each image displays a block of well array corresponding to 10,000 microwells. Scale bar, 200 μm. C , D The ratio of fluorescent beads to trapped beads in a block of well array is plotted as the concentration of Aß captured on CTB-coated beads (CTB-BAN50) in APP-N2a-derived EVs. Plots on the semi-logarithmic ( C ) and linear ( D ) scales are shown. Data represent mean ± SD ( n = 3 each). E Representative images of APP-N2a-derived EVs (2500 ng protein) in the double color idICA using anti-CD9 antibody and BAN50. Each image displays a block of well array corresponding to 10,000 microwells. Scale bar, 200 μm. F The ratio of BAN50 or CD9 fluorescent beads to trapped beads. G The overlap rate between BAN50 and CD9 fluorescent beads. Data represent mean ± SD ( n = 5 each)

Article Snippet: Monoclonal antibodies against CD9 (MAB5218, R&D Systems, Minneapolis, MN), Aß (BAN50, FUJIFILM, Tokyo, Japan), and ßIII tubulin (#2146, Cell Signaling, Danvers, MA) were used.

Techniques: Western Blot, Derivative Assay, Construct, Blocking Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Factor H-related protein 1 (FHR-1) is associated with atherosclerotic cardiovascular disease

doi: 10.1038/s41598-021-02011-w

Figure Lengend Snippet: FHR-1 and FHRE circulate on extracellular vesicles (EVs) in normal human serum (NHS) and normal mouse serum (MS). (A) Detection of FHR-1 in the EV fraction from NHS (EV NHS ) but not from homozygous FHR-1-deficient (EV ΔFHR-1 ) human serum by western blot analysis. FHR-1 was also absent from the supernatant fractions of both sera. Results shown are representative of three experiments. An uncropped gel is shown in . (B) Tracking of EV NHS and EV ΔFHR-1 by live-cell imaging using CLSM. EVs were stained with anti-FHR-1 antibodies conjugated with Alexa Fluor 647 (red). Bars: 10 µm. (C–F) Size distribution and number of vesicles isolated from EV FHR-1 and EV FHRE determined by DLSM using NanoSight NTA 3.2 software. Graphs in ( C,E ) represent overlays of results from 3 to 4 donors. ( (C) , SEM ± standard error, *p ≤ 0.0255 by unpaired two-tailed t-test, n = 4), and ( (E) , SEM ± standard error, *p ≤ 0.0232 by unpaired two-tailed t-test, n = 3). (G) Tracking by live-cell imaging using CLSM of EV FHR-1 and EV ΔFHR-1 stained with anti-CD9 antibody (vesicle marker) and SYTOX orange (nucleic acid marker), Bars: 10 µm. (H) Size distribution of EVs transporting CD9 and FHR-1 (EV CD9&FHR-1 ). EV FHR-1&CD9 were captured with anti-CD9-coated beads from EV FHR-1 , isolated from 1 mL NHS and analyzed by DLSM using NanoSight NTA 3.2 software. (I) Particle numbers (J) and sizes (K) in fractions obtained by size-exclusion chromatography of EV NHS measured by DLSM. (L) High FHR-1 content in fractions 13–16 determined by ELISA. FHR-1 (red) (M) and FHR-1, and vesicle marker CD9, were in close proximity (red) (N) in atherosclerotic tissues, mainly in blood vessels (stippled lines). Complexes were analyzed by proximity ligation assays using anti-FHR-1 and anti-CD9, and were detected by CLSM. EV FHR-1 carry nucleic acids (orange). Bars = 10 µm.

Article Snippet: FHR-1-transporting EVs were captured using beads coated with monoclonal FHR-1 antibody and were stained with Alexa Fluor 647-labeled anti-human CD9 antibody (1:100; Novus Biological, cat no. NB500-327).

Techniques: Western Blot, Live Cell Imaging, Staining, Isolation, Software, Two Tailed Test, Marker, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Ligation

Journal: Scientific Reports

Article Title: Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage

doi: 10.1038/s41598-022-06088-9

Figure Lengend Snippet: Effect of differential centrifugation on EVs using flow cytometry. ( A ) Schematic diagram of differentially processed plasma using single spin (S1: 1000 × g centrifugation) and double spin (S2: 15,000 × g secondary spin after the initial single spin S1). ( B ) Platelet concentration in differentially processed plasma from three healthy individuals (n = 3) was measured in independent technical replicates using a haemocytometer. The error bar represented standard deviations for the indicated blood processing conditions. P -value was calculated using Wilcoxon test (* P < 0.05). ( C ) Representative flow cytometry dot plot of EV diameter (nm) versus fluorescent intensity in Quantum Alexa Fluor MESF units for S1 and S2 using FlowJo. Quantum Alexa Fluor 647 MESF was used for Alexa Fluor 647 conjugated CD9 stained plasma, Quantum Alexa Fluor 488 MESF was used for Alexa Fluor 488 conjugated CD63 stained plasma, and Quantum PE MESF was used for PE conjugated CD41 stained plasma. Events were gated into two subpopulations: 150 to 1000 nm (green box) and from 1000 to 3000 nm (red box).

Article Snippet: CD9 Alexa Fluor 647 (R&D system, clone: #209306, cat. FAB1880R-100 μg) was diluted to a final concentration of 0.001 mg/ml for staining.

Techniques: Centrifugation, Flow Cytometry, Clinical Proteomics, Concentration Assay, Staining

Journal: Scientific Reports

Article Title: Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage

doi: 10.1038/s41598-022-06088-9

Figure Lengend Snippet: Effect of a freeze thaw cycle on EVs using flow cytometry. ( A ) Schematic diagram of differentially processed plasma (S1, S2) and respective freeze thaw processes (S1FR, S2FR). ( B ) Box plot of CD9 + , CD63 + , and CD41 + of gated events from 1000–3000 nm (red) and 150–1000 nm (green) for S1, S1FR, S2, and S2FR using R. CD9 + , CD63 + , CD41 + events were converted to concentrations using calibrated flow rate in a given acquisition time. EV concentration defined as the number of EVs per μl was determined by number of EVs detected in a given sample volume multiplied by the dilution factor. The sample volume was calculated by the product of measured flow rate and acquisition time. Statistical significance were obtained from three healthy volunteers for each freeze thaw processing condition using Tukey’s multiple comparisons (ns = not significant, P > 0.05; * P < 0.05, *** P < 0.001, **** P < 0.0001).

Article Snippet: CD9 Alexa Fluor 647 (R&D system, clone: #209306, cat. FAB1880R-100 μg) was diluted to a final concentration of 0.001 mg/ml for staining.

Techniques: Flow Cytometry, Clinical Proteomics, Concentration Assay

Journal: Scientific Reports

Article Title: Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage

doi: 10.1038/s41598-022-06088-9

Figure Lengend Snippet: Effect of post-thaw processing on EVs using flow cytometry. ( A ) Schematic diagram of differentially processed plasma (S1, S2), respective freeze thaw samples (S1FR, S2FR), and secondary spin after post-freeze/thaw plasma S1FR (S1FRS2). ( B ) Representative flow cytometry dot plot of EV diameter (nm) versus fluorescent intensity in Quantum MESF units for CD9 + EVs, CD63 + , and CD41 + EVs in S1FR, S2FR, and S1FRS2 conditions using FlowJo. Quantum Alexa Fluor 647 MESF is used for Alexa Fluor 647 conjugated CD9 stained plasma, Quantum Alexa Fluor 488 MESF is used for Alexa Fluor 488 conjugated CD63 stained plasma, and Quantum PE MESF is used for PE conjugated CD41 stained plasma. Events were gated from 150 to 1000 nm (green box) and from 1000 to 3000 nm (red box). ( C ) Box plot of CD9 + , CD63 + , CD41 + EV concentration from 1000–3000 nm (red) and 150–1000 nm (green) for S1FR, S2FR, and S1FRS2 using R. Statistical significance were obtained from three healthy volunteers for each freeze thaw processing condition using Tukey’s multiple comparisons (ns = not significant, P > 0.05; * P < 0.05, *** P < 0.001, **** P < 0.0001).

Article Snippet: CD9 Alexa Fluor 647 (R&D system, clone: #209306, cat. FAB1880R-100 μg) was diluted to a final concentration of 0.001 mg/ml for staining.

Techniques: Flow Cytometry, Clinical Proteomics, Staining, Concentration Assay