Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: Flow cytometric characterization of CD81 and LDLr expression on MOLT-4 cells. MOLT-4 cells were grown for 48 h in lipoprotein-rich medium (A) or for 24 h (B) or 48 h (C) in lipoprotein-deficient medium. Background fluorescence was measured with an irrelevant isotype-matched control MAb. CD81 expression was measured with JS64 MAb, and LDLr expression was measured with C7 MAb. Cell-bound antibodies were detected with anti-mouse IgG Oregon green (FITC).

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Expressing, Fluorescence

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: HCV and E2 binding to MOLT-4 cells with low or high levels of LDLr expression

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Binding Assay, Expressing

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: Inhibition of HCV and HCV E2 protein binding to MOLT-4 cells by human soluble CD81, mouse soluble CD81, and LDL

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Inhibition, Protein Binding

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: Specificity of HCV-E2 interactions with human CD81. Soluble human or mouse CD81 (10 μg/ml) applied to nitrocellulose was incubated with HCV-E2 (10 μg/ml). E2 applied to nitrocellulose served as the positive control, and an irrelevant control peptide served as the negative control. Interactions of E2 with each protein were detected with anti HCV-E2 MAb, anti-mouse IgG-AP, and 5-bromo-4-chloro-3-indolyl phosphate–nitroblue tetrazolium substrate.

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Incubation, Positive Control, Negative Control

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: HCV-E2 does not interact with LDL. HCV-E2, HCV NS3/4, human soluble CD81, or mouse soluble CD81 spotted on nitrocellulose were incubated with LDL (10 μg/ml). The positive control consisted of goat-anti LDL polyclonal antibody spotted on nitrocellulose. Interaction of spotted proteins with LDL was detected with mouse anti-LDL MAb and anti-mouse IgG AP followed by 5-bromo-4-chloro-3-indolyl phosphate–nitroblue tetrazolium substrate. Binding was quantitated by densitometry (AlphaImager 2000; Alpha Innotech Corp., San Leandro, Calif.), and results represent density units × 100.

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Incubation, Positive Control, Binding Assay

Journal: Cells

Article Title: Calmodulin as a Key Regulator of Exosomal Signal Peptides

doi: 10.3390/cells12010158

Figure Lengend Snippet: Interaction of CaM with HSP70 and CD81 in T-REx cells ( A ) The peptide solution extracted from immunoprecipitants with the CaM antibody from T-REx cells was analyzed by MALDI-TOF-MS. ( B ) The peak intensity at m/z 861 was measured by MALDI-TOF-MS. ** p < 0.01 vs. Mock cells in the absence of W13 and EGTA. C; control, Mock; T-REx Mock, AspALP; T-REx AspALP. ( C ) Western blot analysis of immunoprecipitants with the CaM antibody from T-REx cells. Representative images from three independent experiments.

Article Snippet: Primary antibodies were directed against CD81 (Novus Biologicals, Centennial, CO) and CD63 (Novus Biologicals) under non-reducing conditions and against CaM (Abcam, Cambridge, UK), Alix (Cell Signaling Technology), HSP70 (Enzo Life Sciences, Farmingdale, NY, USA), and GAPDH (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan).

Techniques: Control, Western Blot

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: Characterization of EV. Particle numbers (A) and relative protein concentrations (B) in the fractions eluted following qEV size-exclusion chromatography. Data are mean ± SEM. (C) Tetraspanin (CD9, CD63, and CD81) content of purified EVs was analyzed in the indicated elution fractions by immunoblotting. (D) Size distribution of pooled fractions 7 to 10 from HDFs (left panel) and L3.6 cells (right panel). (E) Electron microscopy image of EV from HDFs (left panel) and L3.6 cells (right panel).

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Size-exclusion Chromatography, Purification, Western Blot, Electron Microscopy

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: polyP on EV derived from cancer cells mediates ligand binding. (A) The amount of polyP associated with cancer cell–derived EV was estimated by comparison with a standard curve (supplemental Figure 2A). (B) Detection of polyP on L3.6-derived EVs following incubation with buffer (HBS), DNase I and RNase A (D/R), or CIP. polyP of different chain lengths was used to estimate the size of EV-associated polyP. L3.6-derived EVs were preincubated with buffer or E coli PPX, and the binding of PPX-Δ12–AF488 (C), anti-CD81–AF488 (D), and or FXII-AF488 (E) was measured. The number of total EV was determined in scatter mode, and the number of ligand-binding EV was determined in fluorescence mode using a 488-nm excitation laser. The differences in fluorescent particle numbers were compared by estimating the area under each curve (AUC) using GraphPad Prism 8. Bars represent means ± SEM. *** P < .001, 1-way ANOVA with multiple comparisons. L, long; M, medium; S, short.

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Derivative Assay, Ligand Binding Assay, Comparison, Incubation, Binding Assay, Fluorescence

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Cellular response and extracellular vesicles characterization of human macrophages exposed to fine atmospheric particulate matter.

doi: 10.1016/j.envpol.2019.07.101

Figure Lengend Snippet: Fig. 3. Characterization of extracellular vesicles derived from THP-1 exposed to PM2.5-0.3. EVs were obtained from THP-1 exposed 6 h, 24 h or 48 h to 0 (NE), 12.5 or 50 mg/mL of PM2.5-0.3. A) Graphs representing the amounts and the mean of size of EVs measured by nanoparticle tracking analysis. Significant differences were evaluated by Student's T test (*p < 0.01); B) Transmission electronic microscopy images of EVs (bar ¼ 100 nm); C) Western-blot using CD63 and CD81 antibodies on EVs obtained from each condition.

Article Snippet: After blocking, membranes were incubated with anti-CD63 or anti-CD81 primary antibodies (Novus Biologicals, Littleton, CO, USA) at 4 C overnight, washed with PBSTween (0.05% Tween 20), and incubated with a Goat anti-Mouse IgG (HþL) secondary antibody, Horseradish Peroxidase (HRP) conjugated (Life Technologies, ThermoFisher Scientific, Illkirch, France) for 60 min. After washing with PBS-Tween, antigeneantibody reactions were detected with a Chemidoc (Biorad, Marnes la Coquette, France) using the ECL Plus Detection kit (Pierce, ThermoFisher Scientific, Illkirch, France).

Techniques: Derivative Assay, Transmission Assay, Microscopy, Western Blot

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Cellular response and extracellular vesicles characterization of human macrophages exposed to fine atmospheric particulate matter.

doi: 10.1016/j.envpol.2019.07.101

Figure Lengend Snippet: Fig. 6. Characterization of extracellular vesicles derived from primary macrophages exposed to PM2.5-0.3. EVs were obtained from pooled supernatants of primary macrophages isolated from 6 donors and exposed 48 h to 0 (NE) or 12.5 mg/mL of PM2.5-0.3. A) Graph representing the amounts of EVs measured by nanoparticle tracking analysis. Significant differences were evaluated by Student's T test (*p < 0.01); B) Graphical representation of size distribution of EVs produced by non exposed (NE) and exposed (12.5 mg/mL) primary macrophages; C) Western-blot using CD63 and CD81 antibodies on EVs obtained from each condition; D) Transmission electronic microscopy images of EVs (bar ¼ 100 nm except when indicated).

Article Snippet: After blocking, membranes were incubated with anti-CD63 or anti-CD81 primary antibodies (Novus Biologicals, Littleton, CO, USA) at 4 C overnight, washed with PBSTween (0.05% Tween 20), and incubated with a Goat anti-Mouse IgG (HþL) secondary antibody, Horseradish Peroxidase (HRP) conjugated (Life Technologies, ThermoFisher Scientific, Illkirch, France) for 60 min. After washing with PBS-Tween, antigeneantibody reactions were detected with a Chemidoc (Biorad, Marnes la Coquette, France) using the ECL Plus Detection kit (Pierce, ThermoFisher Scientific, Illkirch, France).

Techniques: Derivative Assay, Isolation, Produced, Western Blot, Transmission Assay, Microscopy

Journal: Nature biomedical engineering

Article Title: Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring

doi: 10.1038/s41551-016-0021

Figure Lengend Snippet: ( a ) Western blot analysis of EphA2 expression on EVs from different pancreatic cancer cell lines (BxPC3, MIAPaCa-2, and PANC-1) and a normal pancreas (HPNE) cell line. ( b ) EphA2-EV and ( c ) total EV signal in HPNE and PANC-1 culture over time, respectively; n=3 triplicate samples/time point. ( d ) Quantification of EphA2-EVs and total-EVs (CD81-EphA2-CD9 and CD81-CD63-CD9 nPES assays, respectively), and EphA2-EVs and CD9-EVs (CD9- and CD81-EV-ELISAs) in plasma of NC (n = 10), pancreatitis (n = 10) and pancreatic cancer (n = 10) patients. All data is normalized to the corresponding NC sample data. ( e ) DFM images and ( f ) EphA2-EV area ratios at indicated time points in plasma samples of nude mice following subcutaneous injection without or with PANC-1 human pancreatic cancer cells (2 × 10 6 cells/mouse); n=3 replicates/sample. Data represent means ± SEM; **p<0.01 and ****p < 0.0001 vs. control mice and †††p<0.005 and ††††p<0.0001 vs. pancreatic cancer baseline by two-way ANOVA followed by a Sidak multiple-comparison test.

Article Snippet: Ninety-six-well plates first incubated with anti-human CD81 antibodies (0.2 μg/mL in PBS; R&D Systems, clones #454720) for 12 h at 4 °C.

Techniques: Western Blot, Expressing, Clinical Proteomics, Injection, Control, Comparison

Journal: bioRxiv

Article Title: Tetraspanin positivity as a function of extracellular vesicle size measured by a modified immuno-TEM protocol

doi: 10.1101/2025.03.13.643132

Figure Lengend Snippet: The size distribution of EVs was determined by high-resolution TEM (A), MRPS and nanoFCM (B) and SP-IRIS (C). The inset in (A) shows a representative micrograph. EVs were also purified by density, using iodixanol flotation gradients, and analyzed by western blot (D). The levels of the tetraspanins CD9, CD81, and CD63 were determined by ExoView (E). The X axis separates categories based on the capture antibody, while the different colors represent the results based on fluorophore-labeled detection antibodies. The percentage of CD9-positive EVs was also determined by conventional flow cytometry (F). All EVs in this figure were purified by SEC, except for those in panel (D).

Article Snippet: For TEM EV assays, mouse anti-human CD9 antibody (IgG1κ, 500 µg/mL, H19a, BioLegend) and mouse anti-human CD81 antibody (IgG2b, 500 µg/mL, MAB4615, R&D Systems) were used.

Techniques: Purification, Western Blot, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: Tetraspanin positivity as a function of extracellular vesicle size measured by a modified immuno-TEM protocol

doi: 10.1101/2025.03.13.643132

Figure Lengend Snippet: Experimental procedure followed for labeling and purification of EVs prior to immuno-TEM (A). TEM micrographs of EVs labeled with either an anti-CD9 or an anti-CD81 antibody (B), or an anti-C23 isotype control (C), and treated with either AuNP-AG* (B, C) or AuNP-BSA (B) on a TEM grid. The yellow triangles indicate the presence of AuNPs on the surface of EVs. The percentage of EVs bound to AuNPs for the different antibodies and AuNPs were calculated (D). Error bars correspond to two independent replicates done on different days with different batches of EVs and AuNPs.

Article Snippet: For TEM EV assays, mouse anti-human CD9 antibody (IgG1κ, 500 µg/mL, H19a, BioLegend) and mouse anti-human CD81 antibody (IgG2b, 500 µg/mL, MAB4615, R&D Systems) were used.

Techniques: Labeling, Purification, Control

Journal: bioRxiv

Article Title: Tetraspanin positivity as a function of extracellular vesicle size measured by a modified immuno-TEM protocol

doi: 10.1101/2025.03.13.643132

Figure Lengend Snippet: Representative micrograph of EVs labeled with anti-CD9 (left) or anti-CD81 (right), and treated with AuNP-AG* (A). Yellow triangles indicate bound AuNPs, and numbered labels count small-EVs (green) and large-EVs (brown). The diameter distribution of EVs labeled with each antibody and containing AuNPs on their surface is shown (B), together with the cumulative distribution of the sample (blue line). The percentage of AuNP-labeled EVs is shown in (C), after segregating EVs into two categories based on a diameter cut-off of 100 nm (small EVs: orange; large EVs: green). I.C.: levels in the isotype control (for EVs of any size), representing the background signal of the assay.

Article Snippet: For TEM EV assays, mouse anti-human CD9 antibody (IgG1κ, 500 µg/mL, H19a, BioLegend) and mouse anti-human CD81 antibody (IgG2b, 500 µg/mL, MAB4615, R&D Systems) were used.

Techniques: Labeling, Control