monoclonal antibodies against cd9  (R&D Systems)

Journal: Cancers

Article Title: Molecular Profiling of A549 Cell-Derived Exosomes: Proteomic, miRNA, and Interactome Analysis for Identifying Potential Key Regulators in Lung Cancer

doi: 10.3390/cancers16244123

Figure Lengend Snippet: Physicochemical, morphological, and biochemical characterization of A549 cell-derived exosomes concentrated via ultrafiltration and isolated using the membrane-based affinity binding method (exoEasy Maxi Kit, QIAGEN). ( a ) DLS analysis of exosomes considering the parameter of volume. The curve represents the means from three successive DLS measurements (n = 3, biological replicates). ( b ) Representative zeta-potential distribution of the exosomes isolated via the proposed methodology. The curves obtained by three successive measurements (n = 3, technical replicates) are depicted. ( c – e ) Cryo-TEM analysis of isolated exosomes. Round-shaped exosomes are depicted (black arrows) with distinct exosomal lipid bilayer (white arrows). ( f ) Detection of CD9 and CD63 exosomal markers as well as cytoplasmic marker β-actin via Western blot analysis. The uncropped bolts are shown in . Equal amounts of protein (30 μg) from exosomes and whole cell lysate as calculated by BCA protein assay kit were loaded in wells of 15% SDS-PAGE gel for separation, followed by Western blotting. ( g ) Flow cytometry-based histogram representing the negative control-beads incubated with the anti-CD9 PE antibody without the addition of exosomes (blue curved), as well as the A549 cell-derived exosomes captured by the beads with subsequent incubation with the anti-CD9 antibody (red curve). The dot plots represent the gating strategies for the exosome containing samples and negative control-beads. Data were analyzed using FlowJo (FlowJo™ Software Ashland: Becton, Dickinson and Company, v10.10.0 (2023) .

Article Snippet: The immunoblots were then incubated with the appropriate primary monoclonal antibody against CD9 or CD63 exosomal marker (1:5000 dilution of CD9/60232-1-Ig and CD63/67605-1-lg Monoclonal antibodies, Proteintech Group, Rosemont, IL, USA) overnight at RT.

Techniques: Derivative Assay, Isolation, Membrane, Binding Assay, Zeta Potential Analyzer, Marker, Western Blot, Bicinchoninic Acid Protein Assay, SDS Page, Flow Cytometry, Negative Control, Incubation, Software

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: Fig. 1 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 106 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: Fig. 2 Quantification of GM1-containing EVs Using the idICA. A Western blot analysis of CD9, ganglioside GM1, and ßIII tubulin in N2a cell lysates (1 × 105 cells/lane) and EVs (1 × 107 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: Fig. 3 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 × 105 cells/lane) and EVs (1 × 107 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