mouse anti human cd81  (Proteintech)

Journal: eLife

Article Title: Matrix-associated extracellular vesicles modulate human smooth muscle cell adhesion and directionality by presenting collagen VI

doi: 10.7554/eLife.90375

Figure Lengend Snippet: ( A ) Characterisation of sEVs extracted from VSMC-derived media and ECM fractions. sEVs were sequentially extracted and fractions were collected and analysed by using ExoView. X-axis – capturing spot antibodies (CD63, CD81, CD9, and control MigG1). ( B ) CD63-bead capturing assay shows similar enrichment in CD63+/CD81 + sEVs in the conditioned media and ECM-associated sEVs extracted using 0.5 M NaCl. VSMCs were plated onto gelatin-covered plates for 10 days and media was collected every 3 days. sEVs were extracted in mild (0.5 M NaCl) conditions and media and 0.5 M NaCl fractions were analysed by using CD63-beads capturing assay. ( C, D ) Exogenously added FN-Alexa568 can be detected in the early endosomes and MVBs. VSMC were serum-deprived for 24 hr and then incubated with FN-Alexa568 for 30 min ( C ) or 3 hr ( D ) in M199-0.5% BSA, thoroughly washed and fixed and stained for EEA-1 ( C , green) or CD63 ( D , blue). Size bar, 10 µm. ( E ) Exogenously added FN-Alexa568 can be detected in MVBs. VSMC were incubated with FN-Alexa568 for 3 hr and stained for CD63 which was visualised with anti-mouse-Alexa488 antibody. Size bar, 10 µm, Note the partial colocalisation of FN and CD63. Size bar, 10 µm. ( F , G ) FN can be detected in the CD63 + MVBs in a sparsely growing VSMCs. VSMC were plated at low (5000 cells per well, F ) or high density 20,000 cells per well, ( G ) and were cultured for 24 hr in M199 supplemented with 2.5% sEV-free FBS, fixed and stained for F-actin, FN and CD63. Size bar, 10 µm ( H ) VSMC plating density influences secretion of CD63+/CD81 + sEVs. Cells were plated at the different density and cultured for 24 hr and sEVs in conditioned media were measured by CD63-beads assay. N=1 biological replicates, n=8 technical replicates. Olympus CKX41 inverted microscope equipped with a 20x objective and QImaging QIcam Fast 1394 camera. Size bar, 50 µm (estimated using manufacturer specifications for microscope and camera field of view). ( I ) FN is presented in sEV along with β1 integrin. ( J ) VSMC were plated on the FN-coated dishes and Alexa568-labelled sEV were added to the cell media for 3 hr. Cells were fixed and stained for filopodia marker Myo10 (green) and vinculin (blue). Note perinuclear localisation of internalised sEVs. Size bar, 10 µm. 3D projection. Myo10 staining channel is removed. ( K ) VSMC were plated on the FN-coated dishes pre-coated with Alexa568-labelled sEV and incubated for 24 hr. Cell staining as in . Note even distribution of sEVs across the extracellular matrix and cell area.

Article Snippet: Upon the sectioning, vascular tissues were dried at room temperature for 30 min, fixed and permeabilised in ice-cold acetone for 10 min, incubated in 1% bovine serum albumin (Cat. No. A2153, Sigma-Aldrich) for 1 hr to block non-specific protein binding, stained with unconjugated mouse anti-human CD81 (M38 clone, NBP1-44861, 1:100, Novus Biologicals) and rabbit anti-human fibronectin (F14 clone, ab45688, 1:250, Abcam) primary antibodies and incubated at 4°С for 16 hr.

Techniques: Derivative Assay, Control, Capturing Assay, Incubation, Staining, Cell Culture, Inverted Microscopy, Microscopy, Marker

Journal: eLife

Article Title: Matrix-associated extracellular vesicles modulate human smooth muscle cell adhesion and directionality by presenting collagen VI

doi: 10.7554/eLife.90375

Figure Lengend Snippet: ( A ) Proteomic profiling of pairwise collected carotid atherosclerotic plaques and adjacent intact arterial segments. A Venn diagram shows the number of plaque-specific (n=213) and intact-specific (n=111) proteins as well as the number of proteins which are common for both vascular regions (1368). ( B ) Partial least-squares discriminant analysis indicates clear classification pattern between the carotid plaques (indicated by red triangles) and adjacent intact arterial segments (indicated by blue circles). N=14 patients. ( C ) Volcano plot illustrates that 46 proteins are significantly overexpressed in plaques whilst 13 proteins are significantly upregulated in adjacent intact arterial segments. ( D, E ) Atherosclerotic plaques were co-stained for fibronectin (FN) and sEV marker, CD81. Cell nuclei were counterstained with DAPI. Main figure: x200 magnification, size bar, 50 µm. Box: x400 magnification, size bar, 15 µm. Note an accumulation of FN in the neointima. ( F ) Spatial distribution of FN and CD81 in the neointima. Note high overlap between FN and CD81 in the extracellular matrix. x200 magnification, size bar, 50 µm. Manders’ split colocalisation coefficient for the overlap of FN with CD81 (M1) and CD81 with FN (M2). Neointima region as in E . ( G ) Co-localisation of endothelial cells (stained for CD31/PECAM1, red colour) and sEVs (stained for CD81, green colour). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue colour). Magnification: ×200, scale bar: 50 µm. Note endothelialised (i.e. those covered with CD31-positive cells) segments at the luminal side and abundant CD81 expression both in the neointima and in the tunica media. Endothelial cells (CD31, red colour) are largely co-localised with CD81-positive extracellular vesicles (green colour). Note CD31-positive capillaries in the neointima. ( H, I ) Quantification of FN content in atherosclerotic plaques. Samples were analysed by western blot and band intensity was quantified in ImageJ. Fold change was calculated as the ratio of band intensity in the atherosclerotic plaque to band intensity in the adjacent intact arterial segments normalised to GAPDH. Note that FN content is elevated in atherosclerotic plaques relative to the adjacent intact arterial segments. Paired t-test.

Article Snippet: Upon the sectioning, vascular tissues were dried at room temperature for 30 min, fixed and permeabilised in ice-cold acetone for 10 min, incubated in 1% bovine serum albumin (Cat. No. A2153, Sigma-Aldrich) for 1 hr to block non-specific protein binding, stained with unconjugated mouse anti-human CD81 (M38 clone, NBP1-44861, 1:100, Novus Biologicals) and rabbit anti-human fibronectin (F14 clone, ab45688, 1:250, Abcam) primary antibodies and incubated at 4°С for 16 hr.

Techniques: Staining, Marker, Expressing, Western Blot

Journal: eLife

Article Title: Matrix-associated extracellular vesicles modulate human smooth muscle cell adhesion and directionality by presenting collagen VI

doi: 10.7554/eLife.90375

Figure Lengend Snippet: ( A ) Proteomic analysis of VSMC-derived sEVs and EVs. Venn diagram. N=3 biological replicates. ( B ) Protein enrichment in the EV and sEV proteome. Heat Map. N=3 biological replicates. ( C ) Western blot validation of sEV cargos. EVs and sEVs were isolated from VSMC conditioned media by differential ultracentrifugation and analysed by western blotting. Representative image from N=3 biological replicates. ( D ) Collagen VI was presented in sEVs from conditioned media and in ECM-associated sEVs. sEVs were isolated from conditioned media and 0.5 M NaCl ECM fraction by differential ultracentrifugation and analysed by western blotting. ( E ) Collagen VI is presented on the surface of sEVs. sEVs were analysed by dot-blot in the non-permeabilised or permeabilised (PBS-0.2%-tween20) conditions. Staining intensity was quantified for non-permeabilised (outside) and permeabilised (inside) conditions. Representative from N=2 biological replicates. ( F ) VSMC adhesion is regulated by collagen VI loaded on sEV. FN matrices were incubated with sEV and anti-collagen VI antibody (COLVI IgG, 50 μg/ml) or control IgG (50 μg/ml). Cell adhesion was tracked by using ACEA’s xCELLigence Real-Time Cell Analysis. ANOVA, N=3 biological replicates, ***, p<0.001, ****, p<0.0001. ( G, H, I ) sEVs promote directional VSMC invasion. VSMCs were treated with control siRNA (Scramble) or collagen VI-specific siRNA pools for 24 hr and were seeded to the FN-enriched Matrigel matrix in a μ-Slide Chemotaxis assay and stained with Draq5. Cell tracking was conducted by OperaPhenix microscope for 12 hr and cell invasion parameters were quantified using Columbus, n indicates the total number of tracked cells per condition, Kolmogorov-Smirnov test, *, p<0.05. ( J ) 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 patients.

Article Snippet: Upon the sectioning, vascular tissues were dried at room temperature for 30 min, fixed and permeabilised in ice-cold acetone for 10 min, incubated in 1% bovine serum albumin (Cat. No. A2153, Sigma-Aldrich) for 1 hr to block non-specific protein binding, stained with unconjugated mouse anti-human CD81 (M38 clone, NBP1-44861, 1:100, Novus Biologicals) and rabbit anti-human fibronectin (F14 clone, ab45688, 1:250, Abcam) primary antibodies and incubated at 4°С for 16 hr.

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

Journal: bioRxiv

Article Title: Nanogram of Arg 1 Delivered by extracellular vesicles restore ARG1 activity in a mouse model of ARG1-D and improve lifespan

doi: 10.1101/2025.09.23.677947

Figure Lengend Snippet: Representative image of Cells and STX-Arg1-in EVs probed for exosome markers (CD9, CD81), Arginase 1 (ARG1), cell contamination markers (calnexin, Hsp60) and Golgi marker (GM130).

Article Snippet: Primary antibody used are as following: rabbit anti-human ARG1 (ThermoFisher Scientific, clone 24H4L3, 1:100 dilution), mouse anti-human actin (Novus Biologicals, clone AC-15, 1:20 dilution), rabbit anti-human CD9 (Cell Signaling, clone D9O1A, 1:100 dilution), rabbit anti-human calnexin (Novus Bio, 1:100 dilution), mouse anti-human CD81 (Novus Bio, 1:10 dilution), rabbit anti-HSP60 (R&D system, 1:100 dilution), rabbit anti-human GM130 (R&D system, 1:10 dilution).

Techniques: Marker