Journal: bioRxiv

Article Title: Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI

doi: 10.1101/2023.08.17.551257

Figure Lengend Snippet: A, Proteomic analysis of VSMC-derived sEVs and EV. Venn diagram. N=3. B, Protein enrichment in the EV and sEV proteome. Heat Map. N=3. C, Western blot validation of sEV cargos. EV and sEV were isolated from VSMC’s conditioned media by differential ultracentrifugation and analysed by western blotting. Representative image from N=3. D, VSMC adhesion is regulated by collagen VI loaded to sEV. FN matrices were incubated with sEV and anti-collagen VI antibody (COLVI IgG) or control IgG. Cell adhesion was tracked by using ACEA’s xCELLigence Real-Time Cell Analysis. ANOVA, N=3. E, F, J, sEV promote directional VSMC invasion. VSMCs were treated with control siRNA (Scramble) or collagen VI-specific siRNA pools for 24h and were seeded to the FN-enriched Matrigel matrix in μ-Slide Chemotaxis assay and stained with Draq5. Cell tracking was conducted by OperaPhenix microscope for 12h and cell invasion parameters were quantified using Columbus. Kolmogorov-Smirnov test, *, p<0.05 I, Real-time PCR analysis of expression of CD9, CD63, CD81, COL6A3, EDIL3 and TGFBI in atherosclerotic plaque. *, p<0.05, Paired t-test, N=5.

Article Snippet: Antibodies were CD9 (SA35-08 clone, NBP2-67310, Novus Biologicals), CD63 (BD Pharmingen, 556019), CD81 (BD Pharmingen TM , 555676, B-11, SantaCruz, sc-166029 and M38 clone, NBP1-44861, Novus Biologicals), Syntenin-1 (Abcam, ab133267), Syndecan-4 (Abcam, ab24511), α-Actinin-4 (Abcam, ab108198), fibronectin (Abcam, ab2413, ab6328 [IST-9] (3D matrix staining) and F14 clone, ab45688 (clinical samples analysis)), β1 activating (12G10) antibody was previously described , 4B4 integrin inhibiting antibody (Beckman Coulter, 41116015), vinculin (Sigma, V9264), α5 integrin (P1D6, Abcam, ab78614), Myo10 (Sigma, HPA024223), gelatin-3BP/MAC-2BP (R&D systems, AF2226), EDIL3 antibody (R&D systems, MAB6046), TGFBI (Sigma, SAB2501486), IgG mouse (Sigma PP54), Anti-collagen Type VI antibody, clone 3C4 (Sigma, MAB1944), p34-Arc/ARPC2 antibody (Millipore, #07-227), Cortactin, LGALS3BP (R&D, AF2226), GAPDH (ab139416, Abcam).

Techniques: Derivative Assay, Protein Enrichment, Western Blot, Biomarker Discovery, Isolation, Incubation, Control, Cell Analysis, Chemotaxis Assay, Staining, Cell Tracking Assay, Microscopy, Real-time Polymerase Chain Reaction, Expressing

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 1. Expression of CD9 in mouse testis. A) Characterization of anti-CD9 antibody-purified mouse testis cells using flow cytometry. Unselected (upper row) and anti-CD9 antibody-selected (lower row) testis cells were stained with FITC-conjugated streptavidin, PE-conjugated anti-a6-integrin antibody, or APC-conjugated anti-c-kit antibody, and the fluorescence was compared with that of controls. The dotted lines show the range of fluores- cence in the controls. Note the increased percentages of all three antigens in the anti-CD9 antibody-selected cell population. B) Immunohistological staining of CD9 antigen in mouse testis. Positive cells were found on the basement membrane of seminiferous tubules (arrows). Staining was also observed in the interstitial cells. Bar 5 50 mm (section). Stain: DAB followed by hematoxylin.

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Cytometry, Staining, Membrane

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 2. Expression of CD9 on mouse spermatogonial stem cells. A) Gross and histological appearance of recipient testes after transplantation of LacZ-marked donor testis cells. Left: Testis from a W recipient mouse 2 mo after transplantation of anti-CD9 antibody-selected cells. Middle: Testis from a W recipient mouse 2 mo after transplantation of unselected testis cells. Right: Histological section from a W recipient testis 2 mo after transplantation of anti-CD9-antibody-selected testis cells. Note the normal appearance and organization of spermatogenesis. Stain: X-gal followed by hematoxylin and eosin. B) Enhanced colonization of recipient testis by anti-CD9 antibody-selected mouse testis cells. The degree of coloni- zation in three experiments is indicated by the number of individual blue colonies. The values (mean 6 SEM) for anti-CD9 antibody-selected cells and unselected cells were 5.5 6 0.8 (n 5 16) and 0.8 6 0.2 (n 5 18) cells per 3 3 104 injected cells, respectively. Bars 5 1 mm (testes) and 50 mm (section).

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Transplantation Assay, Staining, Injection

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 3. Expression of CD9 on rat spermatogonial stem cells. A) Characterization of anti-CD9 antibody-purified rat testis cells using flow cytometry. Unselected (left) and anti-CD9 antibody-selected (right) testis cells were stained with Cy5-conjugated anti-mouse IgG antibody, and the fluorescence was compared with similar cell controls. The dotted lines show the control range of fluorescence. Note the increase in percentage of CD9-positive cells after magnetic selection. B) The appearance of recipient testes after transplantation of green fluorescent protein-marked donor testis cells. Seminiferous tubules were dissected using fine forceps. Left: Testis from a busulfan-treated nude recipient mouse 3 mo after transplantation of unselected testis cells (8.2 3 104 cells injected). Right: Testis from a busulfan-treated nude recipient mouse 3 mo after transplantation of anti-CD9 antibody-selected cells (2.7 3 104 cells injected). Note the increase in number of colonies of CD9-positive cells despite the lower concentration of injected cells. Bar 5 100 mm. C) Enhanced colonization of recipient testis by anti-CD9 antibody-selected rat testis cells. The number of individual fluorescent colonies represents the degree of colonization in three experiments. The values (mean 6 SEM) for anti-CD9 antibody-selected and unselected cells were 15.8 6 4.2 (n 5 16) and 3.1 6 0.9 (n 5 13) cells per 105 injected, respectively.

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Cytometry, Staining, Control, Selection, Transplantation Assay, Injection, Concentration Assay

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Overall workflow of EV immunolabelling and Fl‐NTA analysis. Cells are cultured, and EVs are enriched from the cell culture medium. Then polyethylene glycol (PEG) is added to precipitate EVs, followed by centrifugation to isolate EVs. Quantum dots (QDs) are conjugated to anti‐CD9 and anti‐CD63 antibodies for specific labelling of EV surface markers. EVs are immunolabelled with QD conjugated antibodies, excess dyes are removed through additional washing steps. Finally, labelled EVs are resuspended for further analysis. Labelled EVs are analysed using Sc‐/Fl‐NTA. The analysis includes optimisation of labelling conditions, size analysis and comparative studies on different fluorophores, EV markers and cell lines.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Cell Culture, Centrifugation

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Characterisation of EVs derived from A549 cells. (A) Sc‐NTA indicating the size distribution of A549‐derived EVs, with the majority falling within the 30–200 nm range. (B) Western blot demonstrating the enrichment of EV‐specific markers, including CD9, CD63 and Hsp70, in EVs isolated from A549 cells, thereby confirming the successful isolation of EVs. (C) SEM image showing the morphology and size of EVs derived from A549 cells, revealing their characteristic spherical shape and nanoscale size.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Derivative Assay, Western Blot, Isolation

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Optimisation of EV immunolabelling conditions. Evaluated by varying (A) antibody dilution, (B) incubation time and (C) staining volumes. According to Nanodrop measurements, the concentrations of the antibodies were as follows: for anti‐CD9 antibody corresponds to 0.90 ± 0.02 µM, and for anti‐CD63 antibody corresponds to 1.90 ± 0.20 µM. Immunolabelling efficiency is represented as the ratio of the number concentration in fluorescence mode to that in light scatter mode, serving as an indicator of labelling success. The lines represent B‐spline interpolation, applied to the experimental data points to visualise the overall trend in labeling efficiency. Error bars represent SD ( n = 3).

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Incubation, Staining, Concentration Assay, Fluorescence, Labeling

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Comparative analysis of size distribution in scatter and fluorescence mode. The NTA plots show the comparison of size distribution of EVs under different conditions: unlabelled EVs measured in light scatter mode (black line), labelled EVs measured in fluorescence mode (blue line) and labelled EVs measured in light scatter mode (red line). Panels (A–C) represent data for CD9‐QD625, while panels (D–F) represent data for CD63‐QD625. All error bars represent SE ( n = 3). (G, H) Stacked bar plots illustrate the percentage distribution of EV sizes in 50 nm intervals for both fluorescence and scatter modes, emphasising differences in EV subpopulations across the size ranges.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Fluorescence, Comparison

Journal: Journal of Extracellular Biology

Article Title: Quantum Dot‐Based Immunolabelling of Extracellular Vesicles and Detection Using Fluorescence‐Based Nanoparticle Tracking Analysis

doi: 10.1002/jex2.70072

Figure Lengend Snippet: Identification of EVs subpopulations from various cell lines and their relative abundance. (A) Comparative size distribution profiles of CD9‐(+) (black line) and CD63‐(+) (red line) EV populations derived from EA.hy926, THP‐1 and A549 cells, measured in fluorescence mode. Error bars represent SE ( n = 3). (B) Bar graph shows relative abundance of CD9‐positive and CD63‐positive EVs as a percentage, with error bars indicating SD ( n = 3). Note that relative abundance was calculated as the number concentration ratio of FM to LSM.

Article Snippet: To compare between QD and organic dyes, Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD63 antibody (IgG1, clone#: H5C6, Cat#: 353037, BioLegend) and Alexa Fluor 488‐conjugated monoclonal mouse anti‐human CD9 antibody (IgG2B, clone#: 209306, Cat#: FAB1880G, R&D Systems) were also used.

Techniques: Derivative Assay, Fluorescence, Concentration Assay