50-0639-41

antibody from Invitrogen Antibodies

Targeting: CD63 ME491, MLA1, TSPAN30

Provider product page for 50-0639-41

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [7]
Comments [0]
Validations
Flow cytometry [1]
Other assay [9]

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Product number: 50-0639-41 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD63 Monoclonal Antibody (H5C6), eFluor™ 660, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: This H5C6 monoclonal antibody reacts with human CD63, a type III member of the tetraspanin family of transmembrane proteins. CD63 is expressed intracellularly on lysosomes, endosomes, and granules of resting platelets and basophils. However, cell surface expression of CD63 can be detected on activated basophils and platelets, monocytes, macrophages, and granulocytes. This receptor is also expressed on endothelial cells, fibroblasts, and smooth muscle cells. Studies have demonstrated that CD63 associates with integrins (VLA-3 and VLA-6) and TIMP-1 to mediate the allergic response. Applications Reported: This H5C6 antibody has been reported for use in flow cytometric analysis. Applications Tested: This H5C6 antibody has been pre-titrated and tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.5 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. eFluor® 660 is a replacement for Alexa Fluor® 647. eFluor® 660 emits at 659 nm and is excited with the red laser (633 nm). Please make sure that your instrument is capable of detecting this fluorochome. Excitation: 633-647 nm; Emission: 668 nm; Laser: Red Laser. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: H5C6
Vial size: 25 Tests
Concentration: 5 µL/Test
Storage: 4° C, store in dark, DO NOT FREEZE!

CD63 protein structure - 50-0639-41 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 hWJMSC-MVs surface expressed molecules analysis. Flow cytometery analysis showed hWJMSC-MVs were positive for some surface expressed molecules typically expressed by MSCs, such as CD9, CD44, CD63, CD73, and negative for CD34, CD45.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: TIMP-1-specific interaction with CD63. ( A ) Yeast AH109 expressing p53 bait (as a positive control), full-length TIMP-1 (amino acids 1-184) lacking signal peptide (T1), full-length TIMP-2 (T2), N-terminal domain (amino acids 1-125) of TIMP-1 (T1N), or C-terminal domain (amino acids 126-184) of TIMP-1 (T1C) was mated with Y187 expressing large T antigen prey (as a positive control), vector alone (as a negative control), or full-length CD63. Diploids grow on SD/-Leu/-Trp selective media. The specific interaction between bait and prey proteins was detected on quadruple dropout (QDO) media SD/-Leu/-Trp/-His/-Ade containing X-alpha-Gal. ( B ) Anti-CD63 immunoprecipitates of MCF10A cell lysates in the presence of 500 ng/ml biotinylated rTIMP-1 (left and middle panel) or rTIMP-2 (right panel) were analyzed by Western blot analysis with anti-TIMP-1 monoclonal antibody, avidin-HRP, and anti-TIMP-2 polyclonal antibody. ( C ) TIMP-1 overexpressing MCF10A (T29) cell lysates were immunoprecipitated with anti-TIMP-1 polyclonal antibody or anti-CD63 monoclonal antibody, followed by immunoblot analysis using anti-CD63 under nonreducing condition or anti-TIMP-1 antibody under reducing condition, respectively. ( D ) MCF10A cells were grown on the coverslips overnight, blocked with PBS containing 10% horse serum 1% BSA, and co-stained with anti-CD63 Ab/Texas red conjugated secondary Ab (red staining), or with anti-TIMP-1 Ab/FITC conjugated secondary Ab (green staining). Co-localization of TIMP

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CD63 mediates TIMP-1 binding to cell surface and TIMP-1 co-localization with integrin beta1 in human breast epithelial cells. ( A ) MCF10A cell lysates were immunoprecipitated with anti-CD63 antibody, followed by immunoblotting with anti-integrin beta1 or anti-TIMP-1 monoclonal antibody. ( B ) AS-shCont#5 and AS-shCD63#19 cells were cultured on the coverslips overnight and incubated with or without 500 ng/ml TIMP-1 for 30 min. Live cells were co-stained with anti-integrin beta1 Ab/Texas red conjugated secondary Ab (red staining) and anti-TIMP-1 Ab/FITC conjugated secondary Ab (green staining). Colocalization of TIMP-1 and integrin beta1 is shown as yellow staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Downregulation of CD63 expression reverses TIMP-1mediated antiapoptotic activity. ( A ) T29-shCont-P and T29-shCD63-P cells were subjected to immunoblot analysis with an anti-CD63 antibody in a nonreducing condition. The bottom panel shows the beta-actin levels of the same blot reprobed with an anti-human beta-actin antibody. ( B ) Conditioned medium and cell lysates of T29-shCont-P and T29-shCD63-P cells were subjected to immunoblot analysis with an anti-TIMP-1 antibody. The bottom panel shows the beta-actin levels of the same blot reprobed with an anti-human beta-actin antibody. Densitometry analysis of the signals between cellular TIMP-1 and beta-actin was presented by normalizing to the signal ratio in T29-ShCont-P cells. ( C ) Cell lysates (40 mug/lane) of 48 h serum-starved MCF10Aneo (Neo), T29, T29-shCont-P, and T29-shCD63-P cells were subjected to immunoblot analysis with anti-active ERKs (pERK1/2) and anti-ERK1/2 antibodies. Densitometry analysis of the signals between pERK1/2 and ERK1/2 were presented by normalizing to the signal ratio in T29 (left panel) or T29-shCont-P cells (right panel). ( D ) Cell lysates (40 mug/lane) of 48 h serum-starved Neo, T29, T29-shCont-P, and T29-shCD63-P cells were subjected to immunoblot analysis with anti-FAK (pY397) and anti-FAK antibodies. Densitometry analysis of the signals between anti-FAK (pY397) and anti-FAK were presented by normalizing to the signal ratio in T29 (left panel) or T29-shCont-P cells (right panel). ( E ) MCF10A

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Characterization of endothelial colony-forming cells (ECFC)-derived extracellular vesicles (EVs). ( A ) Isolated ECFC-derived EVs were conjugated to CD63-coated latex beads to aid detection by flow cytometry. The gated bead-bound population (left panel) tested positive for EV markers CD9 and CD63. ( B ) Electron micrograph demonstrating the heterogeneity of the EV population (scale bar 1 mum). ( C ) Higher-magnification electron micrograph showing EVs of variable sizes and including a multivesicular body (scale bar 200 nm). ( D-F ) Electron micrographs of EVs bound to latex beads (LB) coated with anti-CD63 antibody. ( D ) White arrows indicate CD63-positive EVs of varying sizes (scale bar 1 mum). ( E ) Higher-magnification micrograph of EVs (scale bar 200 nm). In ( F ), a multivesicular body appears to be encapsulated within a CD63-positive membrane (scale bar 200 nm).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 CTSS is produced in macrophage and transported by exosomes. Western blot analysis of CTSS in macrophage cell lysates and medium after PA (500 muM) stimulation for 24 h ( A ). Western blot analysis of exosome-specific proteins in exosomes and cell lysates detected 24 h after palmitic acid (PA, 500 muM) stimulation of macrophage ( B ). Electron microscopy of exosomes ( C ). (Scale bars = 500 nm) Exosome-derived CTSS from PA (500 muM) stimulated macrophage ( D ). QRT-PCR ( E ) and western blot ( F ) analysis of CTSS gene knockdown efficiency in macrophage. Data are shown as the mean +- SEM, n = 3, ANOVA. * P < 0.05. Detection of CTSS in exosomes from PA (500 muM) stimulated macrophage; macrophage were transfected with shCTSS, scr transfection as the control ( G )

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 M2-TAM-derived exosomes enhance the stemness of GSCs. A TEM images of M2-TAM-derived exosomes. The presence of CD63, CD206, and CD163 in M2-TAM-derived exosomes analyzed by flow cytometric ( B ) and Western blot ( C ) analyses. D Fluorescence images of GSCs co-cultured with Dil-labeled M2-TAM-derived exosomes with nuclei stained with DAPI (blue). E Representative images showing neurosphere formation of GSCs along with the statistics of GSC formation rate and diameter of spheres. F Expression of stem cell-related protein CD133, Nestin, Oct4, Sox2, and GFAP in GSCs measured by Western blot analysis. G HE staining images of xenograft tumors from mice. H The representative immunohistochemical images of Sox2 in xenograft tumors and the percentage of GSCs labeled by Sox2. I Kaplan-Meier survive curve of tumor-bearing mice. n = 10. * p < 0.05 vs. the control EXO group. Measurement data were depicted as mean +- standard deviation, comparison of that between two groups was conducted by unpaired t test. Cell experiments were repeated three times independently.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 ( A ) Flow cytometric data confirmed exosomes by identifying exosomal marker CD63 and 90 +- 8% of exosomes in suspension represented CD63 marker. ( B ) Representative micrograph from transmission electron microscopy showing the nano-sized exosomes (Scale bar 40 nm). Exo: exosomes.