10630D

antibody from Invitrogen Antibodies

Targeting: CD81 TAPA-1, TAPA1, TSPAN28

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Antibody data

Antibody Data
Antigen structure
References [52]
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Validations
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Product number: 10630D - Provider product page
Provider: Invitrogen Antibodies
Product name: CD81 Monoclonal Antibody (M38)
Antibody type: Monoclonal
Antigen: Other
Description: The Exosome - anti-CD81 antibody recognizes human CD81, a member of the tetraspanin family, and is predicted to react with feline and rabbit. The antibody has been verified for western blotting of cellular and exosomal CD81 antigen, with a molecular weight of ~25 kDa, and used at 2 µg/mL under non-reducing conditions. Other applications may work but have not been tested.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: M38
Vial size: 200 µL
Concentration: 0.5 mg/mL
Storage: 4° C

CD81 protein structure - 10630D shown in red.

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

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Experimental details: Figure 1 REG3beta inhibits the uptake of EVs both in vitro and in vivo . ( a ) Transmission electron microscopy images of 120,000 x g pelleted THP1-EVs and MPC-EVs. 2x magnification in the lower right corner to appreciate the double membrane. Scale bars: 200 nm. ( b ) Representative Western blot of EVs samples and cell lysates to confirm the presence of classical exosome markers (CD81, ALIX, TSG101) and the absence of endoplasmic reticulum contamination (Calnexin). ( c,d ) Fluorescence microscopy of THP-1 macrophages ( c ) and MIA PaCa-2 cells (MPC) ( d ) incubated, respectively, with 3 ug/ml of PKH26-labeled MPC EVs or THP1-EVs and increasing concentrations of REG3beta. Nuclei counterstained with DAPI. On the right, quantification of the amount of EVs internalization via fluorimetric reading (n = 4). Data are expressed as mean +- SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, compared to 0 ng/ml REG3beta. ANOVA with Tukey's post-test was used to calculate P -values. Scale bars: 50 um. ( e ) PKH26-labeled EVs injected into tumor xenografts were uptaken by tumor cells (-REG3beta) but remained in the intercellular space when pretreated with REG3beta (+REG3beta). Nuclei counterstained with DAPI. 4x magnification in the top right corner. Scale bars: 50 um.

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Experimental details: Figure 1 Isolation and characterization of exosomes. (A) Image of exosomes observed by transmission electron microscopy. (B) Size distribution of exosomes secreted by non-treated HCT116 cells. (C) Western blot of alpha-tubulin, CD63 and CD81 in exosomes and cells.

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Experimental details: Figure 1 Mesenchymal-epithelial transition factor (MET) expression in AGS cells and cell-derived exosomes. A, Western blotting analysis was used to detect MET protein expression in AGS cells with and without Helicobacter pylori infection for the indicated times. B, qPCR analysis of MET mRNA levels in AGS cells infected with H. pylori for 0, 12 and 24 h. C, The relative expression of Rab27b mRNA was analysed by qPCR in AGS cells infected with H. pylori for 0, 12 and 24 h. * P < 0.05. D, Transmission electron microscopy image of exosomes derived from the AGS cells. Scale bars represent 100 nm. E, Western blotting analysis showing the presence of CD63 and CD81 and the absence of tubulin in AGS cell-derived exosomes. F, MET and p-MET expression in exosomes isolated from AGS cells with and without H. pylori infection was analysed by Western blotting analysis

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Experimental details: Fig 3 Exosome isolation by the size exclusion chromatography. In (a) representative immunoblot showing the distribution of exosome markers (CD63, CD9, CD81) and high-abundance serum proteins (illustrated by Ponceau S staining) in the successive SEC fractions of a FaDU culture medium. In (b) total protein concentrations (mug/uL) in the subsequent SEC fractions. In (c) culture medium supplemented with 5% ED FBS and NOT co-cultured with cells was analyzed as in Panel A; ""E+"" denotes exosome-containing positive control.

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Experimental details: Fig 4 Protein markers of MDM-derived sEVs. (A) Representative image of three independent assays of polyacrylamide gel stained with silver nitrate after separation of 40 mug total protein from cell (Cell) or sEVs pool lysates (EVs; pools comprise samples from four individual donors). (B) Western blot analysis of sEVs markers (Alix, CD63, and CD81) and non-EVs markers (Calnexin and Cytochrome C) ( n = 3). beta-actin = loading control. 40 mug of total protein were loaded onto the gel. MW: molecular weight marker.

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Experimental details: Figure 1 Characterisation of exosome-like vesicles released from serum by ( A ) transmission electron microscopy, ( B ) dynamic light scattering analysis (133+-25 nm) and ( C ) western blotting. Presence of exosomal markers, CD63, CD9, CD81 and mitochondrial protein cytochrome c in lysates from sera-derived exosomes and cell lysate.

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Experimental details: Figure 1 Characterization of exosomes isolated from plasma. ( A ) Representative transmission electron microscopy (TEM) image of exosomes. Scalebar = 200 nm. ( B ) Representative size distribution of exosomes measured by nanoparticle tracking analysis (NTA). ( C ) Exosomes derived from plasma of healthy donors (HD) and head and neck squamous cell carcinoma (HNSCC) patients were analyzed by Western blot for the presence of exosome specific markers using antibodies against CD63 and CD81 under non-reducing conditions and antibodies against CD9 and TSG101 under reducing conditions. Western blot analysis for the negative marker Grp94 and the apolipoprotein Apo1A was also performed for exosomes, cells, and plasma.

Supportive validation

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Experimental details: Figure 1 Characterization of osteosarcoma 143-B-derived extracellular vesicles (EVs) and their uptake by recipient cells. ( A ) 143-B EV size distribution assessed by NanoSight analysis. ( B ) Transmission electron micrographs of 143-B EVs. Scale bar, 250 nm. ( C ) Representative histogram of flow cytometry analysis of exosomal markers CD63 and CD81 in 143-B EV-coated beads. ( D ) Western blot analysis of EV markers (ALIX, CD9) and a cellular marker (calreticulin) in 143-B whole cell protein extracts and in eluted EV fractions from iodixanol based density gradient ultracentrifugation of 143-B EVs. Internalization of PKH67-labelled 143-B EVs by human umbilical vein endothelial cells (HUVEC) ( E ), WI-38 ( F ) and MRC-5 ( G ) cells, as measured by flow cytometry. Representative histograms indicating the percentage of cells internalizing EVs (10 ug) over time (left panels) and the quantification of mean fluorescence intensity (MFI) indicating dose- and time-dependent EV uptake (right panels). The data represent means +- SEM from three independent experiments.

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Experimental details: Fig. 3 Characterization of control EVs and Cur-EVs. a Representative western blot image showing bands of standard surface markers (CD9, CD63, CD81) of control EV- and Cur-EV lysates. b , c Morphology of control EVs and Cur-EVs was monitored by TEM; scale bar, 100 nm. d , e Particle size distribution of control EVs and Cur-EVs was measured by NTA. f , g Quantitative comparison between control EVs and Cur-EVs in count and size measured by NTA; n = 3. h Absorbance at 420 nm of control EVs and Cur-EVs was determined with by spectrophotometry indicating presence of curcumin in Cur-EVs; n = 3. i Cell nuclei were stained with DAPI (blue) and chondrocytes were stained with Phalloidin (green) to visualize the structure of the cytoskeleton. PKH26-labeled control EVs (red) and Cur-EVs (red) were internalized by chondrocytes and visualized with fluorescent microscopy

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Experimental details: Figure 2 Western Blot results. Characterization of isolated extracellular vesicles by Western Blotting analysis using primary antibodies directed to CD81, Alix and loading control beta-actin. Bands were obtained using an exposure of 100 s.

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Experimental details: Fig. 6 Claudin-2 is enriched in extracellular vesicles isolated from patients with replacement-type colorectal cancer liver metastases. a Nanoparticle tracking analysis of EVs detected in plasma derived from either a RHGP liver lesion bearing patient or a DHGP liver lesion bearing patient. b Representative immunoblot analysis of Claudin-2, Claudin-4, CD81, EpCAM, and TSG101 using lysates prepared from EVs isolated from patients with the indicated lesions. c Claudin-2 levels in EVs concentrated from plasma of liver-metastatic CRC patients with the indicated lesions were normalized to TSG101 levels. d Immunoblot analysis of Claudin-2 and TSG101 using lysates prepared from EVs concentrated from patients, pre- and post-resection, with the indicated lesions. e Claudin-2 levels in EVs concentrated from plasma, pre- and post-resection, of liver-metastatic CRC patients with the indicated lesions were normalized to TSG101 levels.

Supportive validation

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Experimental details: Fig. 4 ALIX and CD81 Western Blot analysis. Bands were obtained using an exposure time of 100 s

Supportive validation

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Experimental details: FIGURE 2 Characterization of control EVs and gene analysis of hsa-124-3p in serum-derived EVs and serum. (A) Representative image of Western blotting showed bands of standard surface markers ( CD9 , CD63 , and CD81 ) of EVs and EV-depleted serum. (B) Morphology of EVs was monitored by TEM; scale bar: 100 nm. (C,D) Particle size distribution of EVs was determined by NTA. (E,F) Gene expression of hsa-miR-124-3p in EVs and serum of different samples at different time points. Compared to the control group: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, # Difference between groups: # p < 0.05; ## p < 0.01; #### p < 0.0001; one-way ANOVA with the Newman-Keuls multiple comparison test.

Supportive validation

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Experimental details: Figure 1 Characterization of control EVs and HR-EVs. EVs were processed for western blot, TEM and NTA after isolated from 500mul serum of control and HR groups. (A) The same amount (30mul) of control EVs, HR-EVs and EVs-depleted-Serum were identified using western blot. Representative western blot image showing bands of standard surface markers (CD9, CD63, CD81) of control EVs and HR-EVs. (B, C) Morphology of control EVs and HR-EVs was monitored by TEM; Scale bar: 100 nm. (D, E) Particle size distribution of control EVs and HR-EVs was determined by NTA. Distribution of EVs with a size of 90-200 nm in diameter in both groups. (F, G) Quantitative comparison between control EVs and HR-EVs in count and size measured by NTA; n = 10; All values represent mean +- standard deviation. * p < 0,05, independent two-tailed Student's t -tests.

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Experimental details: Figure 2 HR promoted the concentration of EVs and CD203c + -EVs and CD63 + -EVs derived from serum. 500mul serum from control and different time point HR groups were used to pellet EVs, finally, the EVs were resuspended in 200mul PBS. The total concentration of EVs was determined by a BCA assay. The protein expression levels of CD63, CD203c, CD9 and CD81 in EVs/serum were identified using western blotting. (A) Total concentration of EVs isolated from same volume serum of control and different time point HR groups, n = 10. (B-F) Relative CD63, CD203c, CD9 and CD81 protein expression levels of EVs derived from same volume of serum in control and HR groups, 30mul EVs/lane, n = 5 (G-I) Relative CD63 and CD203c expression levels in same volume of serum in the control and HR groups, 30mul serum/lane, n = 5. All values represent the mean +- standard deviation. Difference to control: ** p < 0,01; *** p < 0,001; # Difference between groups: # p < 0,05; ## p < 0,01; 1 way ANOVA with Newman-Keuls Multiple Comparison Test.

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Experimental details: FIGURE 5 EV characterization and validation. (A) Representative SEM pictures of BMSC EVs. White arrows label EVs with 97 nm (left image) and 115 nm (right image) diameter. Magnification: 60,000x. (B) Representative TEM pictures of BMSC EVs. Scale bar left image = 200 nm, magnification: 100,000x. Right image shows magnification of the EV in the green box. The size of objects was determined (red lines). Vertical line = 104 nm, horizontal line = 113 nm. (C) Western Blot analysis of EV specific surface markers CD9 (left image) and CD81 (right image) in BMSC, CA, CA/OP, and OP EVs and in the EV-depleted FCS depl - uc . Lane 1 = MW ladder, Lane 2 = 5 mug; Lanes 3-6 = 8.2 mug; Lane 7 = 10 mul; Exposure time: left image = 3 min, right image = 10 min (Pierce femto Kit). For respective Ponceau Red images, see Supplementary Figure 3 . (D) Test for EV uptake of BMSC-derived EVs into cultured BMSCs using PKH-26 (red) stained EVs (lower panel). PBS solution + PKH-26 stain was used as negative control (upper panel). Nuclei were counterstained with DAPI. Scale bar 100 mum.

Supportive validation

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Experimental details: Figure 2 Characterization of hBMSC-derived EVs. (A) Representative western blot image ( n = 4) demonstrates standard surface markers (CD9, CD63, and CD81) of hBMSC-derived EVs (lane 1: hBMSC lysate; lane 2: hBMSC-EVs). (B) Particle size distribution of hBMSC-EVs was measured by NTA. (C) Morphology of hBMSC-EVs was monitored by SEM, scale bar: 1 mum. (D) Cell nuclei were stained with DAPI (blue) and chondrocytes were stained with phalloidin (green) to visualize the cytoskeleton. PKH26-labeled hBMSC-derived EVs (red) internalized by chondrocytes were visualized with fluorescent microscopy.

Supportive validation

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Experimental details: Figure 3 Characterization of EV-depleted FCS (FCS depl-uc ). Prior to ultracentrifugation, normal FCS was diluted in culture medium to different concentrations (20, 50%, and undiluted = 100%); different FCS depl-uc groups were identified after ultracentrifugation. (A,B) FCS depl-uc-20% was controlled for EV surface makers (CD9, CD63, and CD81) detected by western blotting (lane 1: hBMSC lysate; lane 2: hBMSC-EVs; lane 3: undepleted FCS; lane 4: FCS depl-uc-20% ). Representative western blot image (A) and Ponceau Red-stained images for each surface marker (B) are shown; n = 3. (C,D) Proliferation and apoptosis of hBMSC were determined by BrdU assay and caspase-3/7 activity assay separately after being incubated in culture medium supplemented with the different FCS groups for 24 h. All values represent mean +- standard deviation. *Significant difference to control: * p < 0.05; ** p < 0.01; # Significant difference between groups: # p < 0.05; ### p < 0.001 one-way ANOVA with Newman-Keuls Multiple Comparison Test; n = 4.

Supportive validation

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Experimental details: Fig. 3 Plasma cell exosomal miR-330-3p could be transferred to ovarian cancer. ( A ) Schematic diagram of plasma cells and ovarian cancer cells cocultured in six-well plates. ( B ) Ovarian cancer cells were cocultured in the absence or presence of primary PKH67-labeled plasma cells (green). Nuclei were stained with DAPI (blue) ( n = 3 for each group). Scale bar, 20 mum. ( C ) Western blot analysis for CD63, CD81, and beta-actin in plasma cell exosomes ( n = 3 for each group). ( D ) Electron micrograph of plasma cell exosomes shows the morphological size (50 to 200 nm). Scale bar, 100 nm. ( E ) Size distribution of exosomes was measured using NanoSight analysis. ( F and G ) Immunofluorescent images of PKH67 abrogation in ovarian cancer cells with respective treatment. Scale bar, 20 mum. Statistical chart was plotted ( n = 3 for each group). ( H ) Scheme chart for small RNA sequencing in plasma cell exosomes in patients with ovarian cancer. tRNA, transfer RNA; rRNA, ribosomal RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; piRNA, Piwi-interacting RNA. ( I ) Venn diagram for overlapped miRNAs identified in ovarian cancer plasma cell exosomes. ( J ) Heatmap for unsupervised hierarchical clustering of GSE73582 dataset using plasma cell exosome-specific miRNA panel as classifiers. ( K ) Cellular programs enriched by GSEA for plasma cell exosome-specific miRNAs represented using Enrichment Map. ( L ) The univariate regression analyses of the identified top miRNAs associa

Supportive validation

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Experimental details: Fig. 5 MAAP promotes association of AAV with EVs. A Schematic of EV isolation by iodixanol density gradient from HEK293 suspension culture. B Immunoblots of iodixanol fractions from suspension cells producing recombinant MAAP8Delta vector complemented in trans with CMV-HA and C CMV-MAAP8-HA. EVs, capsid and MAAP were analyzed from the media of HEK293 producing cells at day 3 post transfection. Capsid and MAAP proteins were analyzed by SDS-PAGE under reducing conditions while EV markers (CD81, CD63, CD9) were analyzed by SDS-PAGE under non-reducing conditions ( n = 2). D Graph displaying percent vector genome titer relative to total viral genomes for each fraction from iodixanol gradient purified MAAP8Delta vector complemented in trans with CMV-HA and CMV-MAAP8-HA. E Schematic of EV isolation by size exclusion chromatography (SEC) from HEK293 suspension culture. F Immunoblots of SEC fractions from suspension cells producing recombinant MAAP8Delta vector complemented in trans with CMV-HA and G CMV-MAAP8-HA. EVs and MAAP were analyzed from the media of HEK293 producing cells at day 3 post transfection. MAAP protein was analyzed by SDS-PAGE under reducing conditions while the EV marker CD63 was analyzed by SDS-PAGE under non-reducing conditions ( n = 2). H Graph displaying percent vector genome titer relative to total viral genomes for each fraction from SEC purified MAAP8Delta vector complemented in trans with CMV-HA and CMV-MAAP8-HA.

Supportive validation

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Experimental details: MuVs and lmEVs present different markers and have different buoyant properties. ( A ) histograms showing the MuV and lmEV particle-size distributions (representative sample, from ALS EVs). ( B ) Representative Western blots showing the detection of CD63, CD81, CD82, AnnexinA1, ARF6, and actin, in MuVs (line1), lmEVs (line 2) and cells (line 3). Exosomal markers were enriched in MuVs and at relatively low or undetectable levels in lmEVs (EVs were extracted from the same cell culture medium for both exosomal and microparticle markers). Protein loaded on the gel is also shown, as loading control. Cellular contamination was not observed as neither of the vesicle fractions were positive for alpha-skeletal actin. ( C ) Vesicle extracts loaded on iodixanol gradients. MuVs presented classic exosomal buoyant properties while the buoyant range of lmEVs extended to a higher iodixanol density. Top panel: representative Western blot showing detection of CD63 for the MuVs at a density of 1.112 g*mL -1 ; bottom panel: representative Western blot (EVs extracted from the same cell culture medium as top panel) showing detection of Annexin A1 and ARF6 for the lmEVs at a density of 1.086-1.112 g*mL -1 and 1.185-1.230 g*mL -1 . Right panel: iodixanol/sucrose density across the 12 fractions; gradient range from 1.048 to 1.263 g*mL -1 . ( D ) Protein concentration in MuVs samples. Protein quantification was below detection sensitivity for lmEVs (ND: not detected). ( E ) Lipid quantification in MuVs

Supportive validation

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Experimental details: Figure 1. Exosomes purified from SACC-83 cells are delivered into HPLF cells. (A) Western blotting analysis showing the presence of CD63, CD81, TSG101 and the absence of GRP94 in SACC-83 cell-derived exosomes. (B) Nanoparticle tracking analysis and (C) transmission electron microscopy images showing exosomes isolated from SACC-83 cells. Magnification, x630; scale bar, 20 um. (D) Confocal microscopy images showing the internalization of PKH67-labeled exosomes in HPLF cells at different incubation times. Scale bar, 5 um. (E) Confocal microscopy images showing the delivery of 0, 25, 50 or 75 ug of exosomes into HPLF cells after 6 h. Scale bar, 5 um. Magnified views are shown on the right. GRP94, glucose-regulated protein 94; HPLF, human periodontal ligament fibroblast; SACC, salivary adenoid cystic carcinoma.