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Fig. 4 Characteristic of EVs derived from UC-MSCs cultured in xeno-free media (UC-MSC-EVs). a Size analysis of EVs using qNano system (Izon Science Ltd). Representative image is shown. b Western blot analysis of selected proteins in UC-MSC-EVs. Three hundred micrograms of protein extracts was used to detect expression of transmembrane (CD63) and cytosolic (syntenin) proteins. Expression of beta-actin was used as control. c Transcript level for extracellular protein (IL-8) measured by RT-qPCR in UC-MSC-EVs. d Surface antigen profile of UC-MSC-EVs by high-sensitivity flow cytometry. The EV samples were stained with the SYTO(r) RNASelect(tm) Green Fluorescent Cell Stain (Molecular Probes) and selected antibodies labeled with a fluorochrome and further analyzed on an A50-Micro Flow Cytometer (Apogee Flow Systems). The percentage of particles positive for indicated surface marker was analyzed from SYTO(r) RNASelect(tm)-positive objects (in gate R1). Representative dot plots for M1-EVs are shown. e Analysis of transcript levels for genes involved in the maintenance of pluripotency ( NANOG ) or differentiation toward cardiac ( GATA4 ) and endothelial lineage ( FLK1 ) performed with the real time PCR method in UC-MSC-EVs. f Relative transcript levels in EVs compared to parental UC-MSCs. Results are shown as mean +- SD. Results were compared with one-way ANOVA and Dunnet's post hoc test, relative to control conditions ( M6 ). * p < 0.05. UC-MSC umbilical cord-derived mesenchymal stem
Figure 3 Survivin is highly enriched in exosomes from PTX-treated cancer cells. ( A ) Western blot analysis using Survivin, flotillin, IkappaBalpha, and CD-63 antibodies was performed on lysates of MDAMB231 cells treated with either DMSO or PTX (lanes labeled WCL), as well as the exosomes (lanes labeled Exos) and MVs (lanes labeled MVs) generated by the cells. ( B ) The relative amounts of Survivin detected in exosomes generated by DMSO- and PTX-treated MDAMB231 cells. ( C ) Western blot analysis using Survivin and actin antibodies was performed on lysates of MDAMB231 cells treated with PTX for increasing lengths of time. ( D ) Immunofluorescence using a Survivin antibody was performed on MDAMB231 cells treated with either DMSO or PTX (top images). The cells were also stained with DAPI to label nuclei (bottom images). Arrows indicate areas where Survivin is detected as puncta in the cytosol of cells treated with PTX. Scale bar = 5 um. ( E ) Western blot analysis was performed using a Survivin antibody on lysates of MDAMB231 cells, U87 glioblastoma cells, and SKBR3 cells that had been treated with DMSO or PTX (lanes labeled WCL), and on the exosomes these cells generated (lanes labeled Exos). ( F ) Western blot analysis was performed on lysates of exosomes from MDAMB231 cells that had been treated with the indicated chemotherapeutic agents and inhibitors. The experiments in B were performed a minimum of three separate times, with each experiment yielding similar results. Stude
Figure 1 MDAMB231 breast cancer cells shed exosomes and exosomes and microvesicles (MVs). ( A ) Outline of procedure used to isolate exosomes and MVs from conditioned medium. ( B ) Western blot analysis using CD-63, IkappaBalpha, and flotillin antibodies was performed on lysates of MDAMB231 cells (lane labeled WCL), and the exosomes (lane labeled Exos), and MVs (lane labeled MVs) that these cells generated. A sample containing all extracellular vesicles (EVs) (including both MVs and exosomes) generated by the cells (lane labeled EVs) was also included on the blot. ( C ) Transmission electron microscopy (TEM) image of exosomes isolated from MDAMB231 cells. Scale bar = 50 nm. ( D ) Histogram showing the sizes of exosomes detected in C. ( E , F ) NIH-3T3 fibroblasts were cultured in serum-free media supplemented without (images and bars labeled Serum Starved) or with either 2% serum (images and bars labeled 2% Serum), or 0.5 x 10 6 exosomes/mL collected from MDAMB231 cells (images and bars labeled Exos) for two days, at which point the cells were stained with DAPI to label nuclei. ( E ) Representative fluorescent images of the nuclei from cells cultured under each of the indicated conditions. Asterisks indicate condensed/blebbed nuclei, a hallmark of apoptosis. Scale bar = 10 mum. The boxed portion of the fibroblasts treated with exosomes (Exos) represents an enlarged image and was placed below the other images to further highlight the differences between a normal (non-apoptotic
Western Blot (WB)
Host / Isotype
Jurkat and HeLa (human cell lines)
PBS, pH 7.4
0.09% sodium azide
Product Specific Information
Tested in western blotting under non-reducing conditions.
CD63 (LAMP-3, lysosome-associated membrane protein-3), a glycoprotein of tetraspanin family, is present in late endosomes, lysosomes and secretory vesicles of various cell types. It is also present in the plasma membrane, usually following cell activation. Hence, it has become an widely used basophil activation marker. In mast cells, however, CD63 exposition does not need their activation. CD63 interacts with integrins and affects phagocytosis and cell migration, it is also involved in H/K-ATPase trafficking regulation of ROMK1 channels. CD63 also serves as a T-cell costimulation molecule. Expression of CD63 can be used for predicting the prognosis in earlier stages of carcinomas.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
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Protein Aliases: CD63; CD63 antigen; CD63 antigen (melanoma 1 antigen); Granulophysin; LAMP-3; Lysosomal-associated membrane protein 3; lysosome-associated membrane glycoprotein 3; ME491; Melanoma-associated antigen ME491; melanoma-associated antigen MLA1; MLA1; Ocular melanoma-associated antigen; OMA81H; Tetraspanin-30; Tspan-30; TSPAN30
Gene Aliases: CD63; LAMP-3; ME491; MLA1; OMA81H; TSPAN30
UniProt ID: (Human) P08962
Entrez Gene ID: (Human) 967
cell adhesion molecule
membrane-bound signaling molecule
Suggested Secondary Antibodies
Material safety data sheets (MSDS)