antihuman anti cd63  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: SIZE DETERMINATION AND MULTIPLEXED FLUORESCENCE-BASED PHENOTYPING OF SINGLE CELL-DERIVED MEMBRANE VESICLES USING A NANOFLUIDIC DEVICE

doi: 10.64898/2026.04.17.719178

Figure Lengend Snippet: Comparison of size distributions and fluorescence-based phenotyping of biomarkers on exosomes using the Nano-SMF method. a) Histogram of CD63-mNeon exosomes and its logNormal fit used to determine mean hydrodynamic diameter and distribution. b) Fluorescence intensity versus hydrodynamic diameter ln-ln plot for the CD63-mNeon exosomes in (a). The line represents the theoretical slope of 2. c) Comparison of normalized fitted size distributions for 50 and 100 nm polystyrene beads (red), LUVs (POPC/RhoB) (green) and CD63-mNeon exosomes (blue). d) Histogram of non-purified, serum fresh, CD63-mCherry exosomes and its lognormal fit. e-h) Subpopulation characterization of CD63-mNeon exosomes (green) labelled with antiCD63 (light blue), the unlabeled CD63-mNeon exosomes in (a) are included as reference (violet). The particles colocalizing the CD63-mNeon and anti-CD63 markers are represented in black. e) Venn diagram of particle subpopulations distribution, the colocalization is represented by the overlapping region. f) Comparison of lognormal fitted size distributions. g) Comparison of the mean hydrodynamic diameters. h) Fluorescence intensity versus hydrodynamic diameter ln-ln plot, showing CD63-mNeon positive particles (green) and CD63-mNeon/anti-CD63 co-localizing particles (black). The line represents the theoretical slope of 2. i-l) Subpopulation characterization of CD63-mNeon exosomes (green) labelled with antiCD81 (red). The unlabeled CD63-mNeon exosomes in (a) are included as reference (violet). The colocalizing particles are represented in black. i) Venn diagram of particle subpopulations distribution, the colocalization is represented by the overlapping region. j) Comparison of lognormal fitted size distributions. k) Comparison of the mean hydrodynamic diameters. l) Fluorescence intensity versus hydrodynamic diameter ln-ln plot showing anti-CD81 positive particles (red) and CD63-mNeon/anti-CD81 co-localizing particles (black). The line represents the theoretical slope of 2.

Article Snippet: Allophycocyanin (APC) conjugated antihuman anti-CD63 and anti-CD81 IgG primary antibodies were purchased from Miltenyi Biotec.

Techniques: Comparison, Fluorescence, Purification

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus.

doi: 10.4049/jimmunol.1301885

Figure Lengend Snippet: FIGURE 3. FasL and TRAIL are enriched on the membrane of placental exosomes. (A) Western blot analyses of FasL and TRAIL protein expres- sion by exosomes from two donors, isolated from supernatants of early placenta explant cultures, compared with expression in early placenta tissue and Jurkat cells. CD63 was used to confirm the exosomal origin of the isolated microvesicles. Protein load, 40 mg/well. (B) Immunoflow cytometry of purified placental exosomes captured on latex microbeads coated with mAbs against FasL and TRAIL and revealed by fluorescence staining with mAbs against the exosomal marker CD63. Superimposed shaded histogram represents negative controls with isotype-matched mAbs. (C) IEM of whole-mount placental exosomes. The typical cup- shaped morphology and size of 30–100 nm is shown by negative contrast staining seen to the left. Specific mAbs and immunogold labeling with 5- or 10-nm gold particles were used to reveal surface protein expression. Anti-CD63 staining confirms the exosomal nature of the isolated micro- vesicles, and their placental origin is revealed by staining for placental alkaline phosphatase (PLAP). FasL staining was performed with 5-nm gold particles and TRAIL with 10-nm particles. Note that in the double staining, FasL and TRAIL are present on separate exosomes. Scale bars, 100 nm.

Article Snippet: Clones and specificities of Abs were as follows: mouse anti-human FasL (G247-4; BD Pharmingen), goat anti-human TRAIL (K-18), mouse antihuman CD63 (MX-49.129.5; Santa Cruz Biotechnology), mouse antihuman pan MIC (clone 6D4; BD Biosciences), FITC-conjugated mouse anti-human CD63 (Immunotech), mouse anti-human CD95 (EOS9.1; eBioscience), mAbs against TRAIL-R1 (clone number 69036) and TRAIL-R2 (clone number 71908) (R&D Systems), CD56 (My31; BD Biosciences), mAbs against CD4, CD8, CD19, and isotype-matched control mAbs IgG1, IgG2a, and IgG2b (DakoCytomation), normal goat serum (Vector Laboratories), and HRP-conjugated secondary Abs (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Western Blot, Isolation, Expressing, Cytometry, Staining, Marker, Labeling, Double Staining