cd81 pe  (Miltenyi Biotec)

Journal: BMC Methods

Article Title: Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach

doi: 10.1186/s44330-025-00034-7

Figure Lengend Snippet: Characterisation of SH-SY5Y cell-derived EVs. SH-SY5Y cells were incubated in SFM supplemented with NGF (50 ng/ml) for 24 h and EVs were isolated from the cleared media using ExoQuick-TC. A The size distribution of EVs was analysed by NTA ( n =4 ± SD). B & C Negative staining of EVs and TEM analysis revealed the presence of EVs with a cup-shaped morphology. Scale bar = 100 nm ( B ) and 200 nm ( C ). D - G EVs were lysed in RIPA buffer and the presence of CD81 ( D ), Tsg101 ( E ), NCAM ( F ) and calnexin ( G ) was analysed by immunoblot analysis (data shows SH-EVs isolated from three T75 flasks of SH-SY5Y cells)

Article Snippet: Exosome-Streptavidin Isolation/Detection reagent Dynabeads (Thermo Fisher Scientific) (100 μl) were conjugated to 1 μg of biotinylated antibodies against L1CAM (clones 5G3 or UJ127; Thermo Fisher Scientific), NCAM (clone CMSSB; Thermo Fisher Scientific), CD81 (clone REA513; Miltenyi Biotec Ltd, Surrey, UK), calnexin (clone CANX/1543; Generon Ltd, Slough, UK), or mouse IgG (Thermo Fisher Scientific) in 100 μl PBS containing 0.1% (w/v) BSA for 1 h at room temperature.

Techniques: Derivative Assay, Incubation, Isolation, Negative Staining, Western Blot

Journal: BMC Methods

Article Title: Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach

doi: 10.1186/s44330-025-00034-7

Figure Lengend Snippet: Flow cytometry of CD81-positive SH-SY5Y EVs immunocaptured using pre-conjugated anti-CD81 Dynabeads. A Schematic representation of an EV captured by an anti-CD81-conjugated immunocapture bead and labelled with anti-CD81-PE. B Increasing volumes (12.5–100 µl) of SH-SY5Y cell-derived EVs were incubated overnight with pre-conjugated anti-CD81 Dynabeads and then labelled with anti-CD81-PE or IgG-PE antibodies. Negative control samples included unlabelled beads or anti-calnexin conjugated magnetic beads incubated with 100 µl of SH-SY5Y EVs. Samples were examined by flow cytometry and median fluorescence intensities were plotted ( n =3 ± SD, One Way ANOVA). C Histogram plots showing anti-CD81-PE fluorescence intensities against bead count. Histograms for control beads (beads without conjugated antibody and anti-calnexin beads) and IgG-PE labelled samples are shown on the left side of the plot. Histograms for anti-CD81-PE labelled samples are shown on the right side of the plot: grey histogram = 100 µl of EVs, yellow histogram = 50 µl of EVs, purple histogram = 25 µl pf EVs and dark green histogram = 12.5 µl of EVs

Article Snippet: Exosome-Streptavidin Isolation/Detection reagent Dynabeads (Thermo Fisher Scientific) (100 μl) were conjugated to 1 μg of biotinylated antibodies against L1CAM (clones 5G3 or UJ127; Thermo Fisher Scientific), NCAM (clone CMSSB; Thermo Fisher Scientific), CD81 (clone REA513; Miltenyi Biotec Ltd, Surrey, UK), calnexin (clone CANX/1543; Generon Ltd, Slough, UK), or mouse IgG (Thermo Fisher Scientific) in 100 μl PBS containing 0.1% (w/v) BSA for 1 h at room temperature.

Techniques: Flow Cytometry, Derivative Assay, Incubation, Negative Control, Magnetic Beads, Fluorescence, Control

Journal: BMC Methods

Article Title: Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach

doi: 10.1186/s44330-025-00034-7

Figure Lengend Snippet: Flow cytometry of CD81-positive SH-SY5Y EVs immunocaptured using anti-NCAM or anti-L1CAM Dynabeads. A Schematic representation of an EV captured by an anti-NCAM/CD81/IgG-conjugated immunocapture bead and labelled with anti-CD81-PE. B Forward and side scatter plot of Dynabeads. C SH-SY5Y cell-derived EVs (100 µl) were incubated overnight with antibody-conjugated beads and then labelled with anti-CD81-PE or IgG-PE antibodies and analysed by flow cytometry ( n =3 ± SD, One Way ANOVA). Histograms are shown for anti-NCAM beads ( D ), IgG beads ( E ) or anti-NCAM beads washed with 0.25 % TX-100 ( F ) and then labelled with anti-CD81-PE (green histograms) or IgG-PE (orange histograms) compared to control beads (without conjugated antibody) labelled with anti-CD81-PE (blue histograms) or IgG-PE (red histograms)

Article Snippet: Exosome-Streptavidin Isolation/Detection reagent Dynabeads (Thermo Fisher Scientific) (100 μl) were conjugated to 1 μg of biotinylated antibodies against L1CAM (clones 5G3 or UJ127; Thermo Fisher Scientific), NCAM (clone CMSSB; Thermo Fisher Scientific), CD81 (clone REA513; Miltenyi Biotec Ltd, Surrey, UK), calnexin (clone CANX/1543; Generon Ltd, Slough, UK), or mouse IgG (Thermo Fisher Scientific) in 100 μl PBS containing 0.1% (w/v) BSA for 1 h at room temperature.

Techniques: Flow Cytometry, Derivative Assay, Incubation, Control

Journal: BMC Methods

Article Title: Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach

doi: 10.1186/s44330-025-00034-7

Figure Lengend Snippet: Flow cytometry of NCAM-positive SH-SY5Y EVs immunocaptured using anti-NCAM Dynabeads. A Schematic representation of an EV captured by an anti-NCAM/CD81/IgG-conjugated immunocapture bead and labelled with anti-NCAM-PE. B SH-SY5Y cell-derived EVs (100 µl) were incubated overnight with antibody-conjugated Dynabeads and then labelled with anti-NCAM-PE or IgG-PE antibodies and analysed by flow cytometry ( n =3/4 ± SD, One Way ANOVA). Histograms of anti-NCAM Dynabeads ( C ) or IgG Dynabeads ( D ) labelled with anti-NCAM-PE (green histogram) or IgG-PE (orange histogram) compared to control beads (without conjugated antibody) labelled with anti-NCAM-PE (blue histogram) or IgG-PE (red histogram)

Article Snippet: Exosome-Streptavidin Isolation/Detection reagent Dynabeads (Thermo Fisher Scientific) (100 μl) were conjugated to 1 μg of biotinylated antibodies against L1CAM (clones 5G3 or UJ127; Thermo Fisher Scientific), NCAM (clone CMSSB; Thermo Fisher Scientific), CD81 (clone REA513; Miltenyi Biotec Ltd, Surrey, UK), calnexin (clone CANX/1543; Generon Ltd, Slough, UK), or mouse IgG (Thermo Fisher Scientific) in 100 μl PBS containing 0.1% (w/v) BSA for 1 h at room temperature.

Techniques: Flow Cytometry, Derivative Assay, Incubation, Control

Journal: BMC Methods

Article Title: Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach

doi: 10.1186/s44330-025-00034-7

Figure Lengend Snippet: Immunoblot and SEM analysis of SH-SY5Y EVs immunocaptured on anti-NCAM beads. A SH-SY5Y cell-derived EVs (100 µl) were incubated overnight with antibody-conjugated beads. The beads were then washed and the immunocaptured EVs lysed in RIPA buffer. Proteins were separated by SDS-PAGE and analysed by immunoblot for Tsg101, NCAM, L1CAM and calnexin. The numbers below the blots indicate the antibodies conjugated to the beads as follows; 1= anti-L1CAM clone 5G3, 2 = anti-L1CAM clone UJ127, 3 = anti-NCAM clone CMSSB, 4 = beads without antibody, 5 = anti-calnexin, 6 = anti-CD81, 7 = SH-SY5Y EVs, 8 = SH-SY5Y cell lysate. Gels are representative of three experiments. B SH-EVs were incubated with anti-NCAM or IgG immunocapture beads overnight. Beads were washed to remove non-bound EVs, and beads were fixed in 2 % PFA and imaged by SEM. Arrows indicate vesicle structures on the surface of the beads. Images shown are at x30,000 (upper images) and x50,000 magnification (lower images)

Article Snippet: Exosome-Streptavidin Isolation/Detection reagent Dynabeads (Thermo Fisher Scientific) (100 μl) were conjugated to 1 μg of biotinylated antibodies against L1CAM (clones 5G3 or UJ127; Thermo Fisher Scientific), NCAM (clone CMSSB; Thermo Fisher Scientific), CD81 (clone REA513; Miltenyi Biotec Ltd, Surrey, UK), calnexin (clone CANX/1543; Generon Ltd, Slough, UK), or mouse IgG (Thermo Fisher Scientific) in 100 μl PBS containing 0.1% (w/v) BSA for 1 h at room temperature.

Techniques: Western Blot, Derivative Assay, Incubation, SDS Page

Journal: BMC Methods

Article Title: Examination of the enrichment of neuronal extracellular vesicles from cell conditioned media and human plasma using an anti-NCAM immunocapture bead approach

doi: 10.1186/s44330-025-00034-7

Figure Lengend Snippet: Characterisation of plasma-derived EVs. EVs were isolated from 0.5 ml of plasma using Exo-spin size exclusion chromatography columns and concentrated using 10 kDa MWCO centrifugal filters. A The size distribution of the EVs was analysed by NTA (average data from three donors ± SD). B TEM of negatively stained plasma-derived EVs revealed the presence of EVs with a cup-shaped morphology (scale bar = 100 nm). EVs from three donors were lysed in RIPA buffer and analysed by SDS-PAGE and immunoblot analysis for CD81 ( C ), CD63 ( D ), Tsg101 ( E ), alpha-tubulin ( F ), calnexin ( G ) and ApoAI ( H )

Article Snippet: Exosome-Streptavidin Isolation/Detection reagent Dynabeads (Thermo Fisher Scientific) (100 μl) were conjugated to 1 μg of biotinylated antibodies against L1CAM (clones 5G3 or UJ127; Thermo Fisher Scientific), NCAM (clone CMSSB; Thermo Fisher Scientific), CD81 (clone REA513; Miltenyi Biotec Ltd, Surrey, UK), calnexin (clone CANX/1543; Generon Ltd, Slough, UK), or mouse IgG (Thermo Fisher Scientific) in 100 μl PBS containing 0.1% (w/v) BSA for 1 h at room temperature.

Techniques: Clinical Proteomics, Derivative Assay, Isolation, Size-exclusion Chromatography, Staining, SDS Page, Western Blot