human cd63 gfp  (Addgene inc)

Journal: Science Advances

Article Title: High-throughput single-EV liquid biopsy: Rapid, simultaneous, and multiplexed detection of nucleic acids, proteins, and their combinations

doi: 10.1126/sciadv.abc1204

Figure Lengend Snippet: ( A ) Illustration of the HNCIB system for simultaneous detection of PD-L1 membrane protein and mRNA in a single EV. Photo credit: Ming Wang, Hangzhou Dixiang Co. Ltd., Hangzhou, China. ( B ) Electron micrograph showing the EVs isolated from human plasma. ( C ) Particle size and zeta potential measurement of the EVs isolated from human plasma by nanoparticle tracking analysis technique. d.nm, diameter (nm). ( D ) Bright-field TIRFM images showing the EVs isolated from human plasma. ( E ) EV marker CD9 and CD63 ( F ) membrane protein measured by the HNCIB system. ( G ) Non-EV markers ALB and APOB ( H ) protein measured by the HNCIB system.

Article Snippet: Lentiviral vector overexpressing human CD63 (CD63-GFP) (GenBank, NM_001267698.1), overexpressing human PD-L1 (GenBank, NM_014143.3), or empty vector was purchased from GeneCopoeia (Guangzhou, China).

Techniques: Membrane, Isolation, Clinical Proteomics, Zeta Potential Analyzer, Marker

Journal: Science Advances

Article Title: High-throughput single-EV liquid biopsy: Rapid, simultaneous, and multiplexed detection of nucleic acids, proteins, and their combinations

doi: 10.1126/sciadv.abc1204

Figure Lengend Snippet: ( A ) Fluorescence signals of PBS only (no EVs and no antibodies), EV only (EVs without staining with any antibodies), EV isotype (EVs stained with isotype control antibody), and EV CD63 antibodies (Abs) (EVs stained with CD63 antibody). ( B ) Fluorescence signals of PBS only, EV only, EV isotype, and EV PD-L1 Abs (EVs stained with PD-L1 antibody). ( C ) Particle size and zeta potential measurement of the EVs isolated from A549 cells overexpressed with CD63-GFP by nanoparticle tracking analysis technique. d.nm, diameter (nm). ( D ) EVs isolated from human A549 cells overexpressed with CD63-GFP or PD-L1–mCherry ( E ) and diluted to different concentrations. 1:1, initial concentration; 1:10, 10-fold dilution; 1:100, 100-fold dilution; 1:1000, 1000-fold dilution.

Article Snippet: Lentiviral vector overexpressing human CD63 (CD63-GFP) (GenBank, NM_001267698.1), overexpressing human PD-L1 (GenBank, NM_014143.3), or empty vector was purchased from GeneCopoeia (Guangzhou, China).

Techniques: Fluorescence, Staining, Control, Zeta Potential Analyzer, Isolation, Concentration Assay

Journal: Science Advances

Article Title: High-throughput single-EV liquid biopsy: Rapid, simultaneous, and multiplexed detection of nucleic acids, proteins, and their combinations

doi: 10.1126/sciadv.abc1204

Figure Lengend Snippet: ( A ) EVs isolated from 1000 μl of cell supernatant of human A549 cells overexpressed with CD63-GFP and PD-L1–mCherry. The dots in the colocalized only image represent EVs expressing both CD63 and PD-L1 protein. ( B ) EVs isolated from 90 μl of human plasma are stained with CD63-AF488 and PD-L1–AF647 antibodies. The dots in the colocalized only image represent EVs expressing both CD63 and PD-L1 protein. ( C ) Ratio of CD63- and PD-L1–positive EVs to CD63-positive EVs isolated from human A549 cells overexpressed with CD63-GFP and PD-L1–mCherry or isolated from plasma of 20 patients with LUAD.

Article Snippet: Lentiviral vector overexpressing human CD63 (CD63-GFP) (GenBank, NM_001267698.1), overexpressing human PD-L1 (GenBank, NM_014143.3), or empty vector was purchased from GeneCopoeia (Guangzhou, China).

Techniques: Isolation, Expressing, Clinical Proteomics, Staining

Journal: Science Advances

Article Title: High-throughput single-EV liquid biopsy: Rapid, simultaneous, and multiplexed detection of nucleic acids, proteins, and their combinations

doi: 10.1126/sciadv.abc1204

Figure Lengend Snippet: ( A ) Double staining with CD63-AF488 and PD-L1–AF647 fluorescent antibodies of EVs in plasma from healthy donors and patients with LUAD. ( B ) EVs in plasma samples were captured with CD63 exosome capture beads and incubated with CD63-AF488 antibody, and FCM was used to detect the expression of EV membrane protein CD63. ( C ) EVs in plasma samples were captured with CD63 exosome capture beads and incubated with PD-L1–AF647 antibody, and FCM was used to detect EV membrane protein PD-L1 expression. ( D ) Statistic analysis of the differential membrane protein expression of PD-L1 + EVs in plasma from three healthy donors and six patients with LUAD. ( E ) Statistic analysis of the differential mRNA expression of PD-L1 in EVs from plasma of six healthy donors and six patients with LUAD.

Article Snippet: Lentiviral vector overexpressing human CD63 (CD63-GFP) (GenBank, NM_001267698.1), overexpressing human PD-L1 (GenBank, NM_014143.3), or empty vector was purchased from GeneCopoeia (Guangzhou, China).

Techniques: Double Staining, Clinical Proteomics, Incubation, Expressing, Membrane

Journal: bioRxiv

Article Title: Harnessing Extracellular Vesicles for Stabilized and Functional IL-10 Delivery in Macrophage Immunomodulation

doi: 10.1101/2025.01.14.633016

Figure Lengend Snippet: a) Size distribution and concentration were measured using nanoparticle tracking analysis. b) Western blot analysis of IL-10 protein showing multiple bands and EV markers CD9, CD63, and CD81. c) IL-10 mRNA expression analyzed by qPCR. d) Proteinase K protection assay for IL-10 + sEVs, with TSG101 serving as a luminal protein control. e) Stability comparison between sEV-incorporated IL-10 and recombinant human IL-10 protein (RP) under 2 h at room temperature (RT), 2 h at 37 °C (37), and 2 cycles of freezing and thawing (F/T). Fresh samples served as controls. Naïve sEVs were isolated from non-transfected HEK293FT cells.

Article Snippet: Human CD63-GFP (Addgene plasmid #62964, gift from Paul Luzio) was subcloned into the pKT2/CAGXSP vector using recombination cloning (In-Fusion HD Cloning Kit, Clontech), as described before [ , ].

Techniques: Concentration Assay, Western Blot, Expressing, Control, Comparison, Recombinant, Isolation, Transfection

Journal: bioRxiv

Article Title: Harnessing Extracellular Vesicles for Stabilized and Functional IL-10 Delivery in Macrophage Immunomodulation

doi: 10.1101/2025.01.14.633016

Figure Lengend Snippet: a) The isolated sEVs underwent a second purification step based on their surface charge by anion exchange chromatography with a linear NaCl gradient (30-500 mM) (Created with BioRender.com ). b) Representative transmission electron microscopy images of sEVs in Protein-high (left), Protein-low (middle), and Flow-through (right) fractions purified from IL-10 + sEVs. c) Western blot analysis of IL-10 protein and Exo markers CD9, CD63 and CD81 in Protein-high fractions 9-12. d) Western blot analysis comparing IL-10 protein expression between naïve and IL-10 + Exos. e) qPCR analysis of pro-inflammatory mRNA markers in LPS-stimulated THP-1 macrophages following 24 h of incubation with 10 µg of Exos.

Article Snippet: Human CD63-GFP (Addgene plasmid #62964, gift from Paul Luzio) was subcloned into the pKT2/CAGXSP vector using recombination cloning (In-Fusion HD Cloning Kit, Clontech), as described before [ , ].

Techniques: Isolation, Purification, Chromatography, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Incubation

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Validation of inhibitors and activators of exosome biogenesis using the qHTS and the quantitative qNano IZON particle analysis: ( A ) Scatter plot of the % efficacy (the difference in maximum and minimum activity in the dose-response) of all the compounds with activity in the qHTS (CRC ±1, ±2, ±3). Highlighted in red are compounds validated to increase CD63-GFP signal and, in blue, those validated to decrease CD63-GFP. ( B ) CRC curves for 22 compounds of the 22 hits obtained from the qHTS assay are shown here. ( C ) C4-2B-CD63-GFP cells were maintained overnight in exosome free RPMI media and then treated with the qHTS hits (compounds) or DMSO (vehicle control) for 48 hours. Treatments were at concentrations of 1 μM for neticonazole and tipifarnib and 10 μM for all other compounds. The exosomes in the conditioned media were isolated, filtered thorough 0.22 μM filter and analyzed by the qNano IZON system using NP-100 nanopore. A detailed description of the protocol is described in “Materials and Methods” section. qNano IZON particle quantitative analysis (NP-100 nanopore) depicting a significant decrease in exosome concentrations (50–200 nm size) in the CM of C4-2B-CD63-GFP cells treated with validated compounds compared to vehicle-treated controls.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Activity Assay, Isolation

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Validation of inhibitors of exosome biogenesis using the MACSQuant Analyzer 10 Flow Cytometer: The exosomes in the conditioned media were isolated and analyzed by the MACSQuant Analyzer 10 Flow Cytometer as described in the “Materials and Methods” section. Treatments were at concentrations of 1 μM for neticonazole and tipifarnib and 10 μM for all other compounds. All the compounds identified as inhibitors using the qNano IZON were validated as potent inhibitors of exosome biogenesis by the MACSQuant, as well (except pentetrazol). The data is represented as the percentage of exosomes and has been normalized to the control sample (100%). Neticonazole (19%), tipifarnib (30%), isoproterenol (49.4%), mitotane (50.7%), pentetrazol (85%) and climbazole (88%) are potent inhibitors of exosome biogenesis. Three controls were used in this experiment. PBS without EVs was used as a reference control. The NO-FL-CONTROL are exosomes from parental C4-2B cells that did not express CD63-GFP cells. Exosomes from DMSO treated C4-2B-CD63-GFP is used as GFP control and PBS served as a reference control. Three samples determined to be activators of exosome biogenesis by the qNano-IZON were also tested by the MACSQuant Analyzer validated to be activators (Fig. ). ***Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Flow Cytometry, Isolation

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Azoles modulate exosome biogenesis through ESCRT dependent and independent pathway. ( A ) C4-2B-CD63-GFP were maintained overnight in exosome free DMEM media and then treated with the azoles or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the NanoFACS system as described in the “Materials and Methods” section. The concentration of exosomes from neticonazole and climbazole -treated cells was significantly lower than DMSO (vehicle) while the concentration of exosomes from nitrefazole treated cells was significantly higher. PBS without EVs was used as a reference control. ( B ) Neticonazole significantly inhibited the protein concentration of Alix, nSMase2 and Rab27a in C4-2B cells and climbazole significantly inhibited the protein concentration of Alix, and Rab27a but not nSMase2. Nitrefazole and pentetrazol showed a significant increase in Alix and nSMase2, but not in Rab27a. Full blots are presented in Supplementary Fig. . ( C ) Neticonazole significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B. Full blots are presented in Supplementary Fig. . ( D ) Neticonazole significantly inhibited the concentration of exosomes measured by qNano IZON in a dose-dependent manner in C4-2B cells. ( E ) Neticonazole significantly inhibited the protein concentration of Alix and nSMase2 in a dose-dependent manner in C4-2B cells. Full blots are presented in Supplementary Fig. . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Ketoconazole (prototype imidazole) inhibits exosome biogenesis through ESCRT dependent and independent pathway. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with ketoconazole or DMSO (vehicle control) for 48 hours at 5 μM. Concentration of exosomes was measured by qNano IZON. Ketoconazole shows a significant decrease in the concentration of exosomes in C4-2B cells at 5 μM as well as in the PC-3 cells ( B ) Ketoconazole significantly inhibited the protein concentration of Alix, nSMase2 and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. . ( C ) Ketoconazole significantly inhibited activation p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B, and PC-3 cells, but not in the RWPE-1 cells. Full blots are presented in Supplementary Fig. . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Validation of inhibitors and activators of exosome biogenesis using the qHTS and the quantitative qNano IZON particle analysis: ( A ) Scatter plot of the % efficacy (the difference in maximum and minimum activity in the dose-response) of all the compounds with activity in the qHTS (CRC ±1, ±2, ±3). Highlighted in red are compounds validated to increase CD63-GFP signal and, in blue, those validated to decrease CD63-GFP. ( B ) CRC curves for 22 compounds of the 22 hits obtained from the qHTS assay are shown here. ( C ) C4-2B-CD63-GFP cells were maintained overnight in exosome free RPMI media and then treated with the qHTS hits (compounds) or DMSO (vehicle control) for 48 hours. Treatments were at concentrations of 1 μM for neticonazole and tipifarnib and 10 μM for all other compounds. The exosomes in the conditioned media were isolated, filtered thorough 0.22 μM filter and analyzed by the qNano IZON system using NP-100 nanopore. A detailed description of the protocol is described in “Materials and Methods” section. qNano IZON particle quantitative analysis (NP-100 nanopore) depicting a significant decrease in exosome concentrations (50–200 nm size) in the CM of C4-2B-CD63-GFP cells treated with validated compounds compared to vehicle-treated controls.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Activity Assay, Isolation

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Validation of inhibitors of exosome biogenesis using the MACSQuant Analyzer 10 Flow Cytometer: The exosomes in the conditioned media were isolated and analyzed by the MACSQuant Analyzer 10 Flow Cytometer as described in the “Materials and Methods” section. Treatments were at concentrations of 1 μM for neticonazole and tipifarnib and 10 μM for all other compounds. All the compounds identified as inhibitors using the qNano IZON were validated as potent inhibitors of exosome biogenesis by the MACSQuant, as well (except pentetrazol). The data is represented as the percentage of exosomes and has been normalized to the control sample (100%). Neticonazole (19%), tipifarnib (30%), isoproterenol (49.4%), mitotane (50.7%), pentetrazol (85%) and climbazole (88%) are potent inhibitors of exosome biogenesis. Three controls were used in this experiment. PBS without EVs was used as a reference control. The NO-FL-CONTROL are exosomes from parental C4-2B cells that did not express CD63-GFP cells. Exosomes from DMSO treated C4-2B-CD63-GFP is used as GFP control and PBS served as a reference control. Three samples determined to be activators of exosome biogenesis by the qNano-IZON were also tested by the MACSQuant Analyzer validated to be activators (Fig. ). ***Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Flow Cytometry, Isolation

Journal: Scientific Reports

Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

doi: 10.1038/s41598-018-26411-7

Figure Lengend Snippet: Azoles modulate exosome biogenesis through ESCRT dependent and independent pathway. ( A ) C4-2B-CD63-GFP were maintained overnight in exosome free DMEM media and then treated with the azoles or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the NanoFACS system as described in the “Materials and Methods” section. The concentration of exosomes from neticonazole and climbazole -treated cells was significantly lower than DMSO (vehicle) while the concentration of exosomes from nitrefazole treated cells was significantly higher. PBS without EVs was used as a reference control. ( B ) Neticonazole significantly inhibited the protein concentration of Alix, nSMase2 and Rab27a in C4-2B cells and climbazole significantly inhibited the protein concentration of Alix, and Rab27a but not nSMase2. Nitrefazole and pentetrazol showed a significant increase in Alix and nSMase2, but not in Rab27a. Full blots are presented in Supplementary Fig. . ( C ) Neticonazole significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B. Full blots are presented in Supplementary Fig. . ( D ) Neticonazole significantly inhibited the concentration of exosomes measured by qNano IZON in a dose-dependent manner in C4-2B cells. ( E ) Neticonazole significantly inhibited the protein concentration of Alix and nSMase2 in a dose-dependent manner in C4-2B cells. Full blots are presented in Supplementary Fig. . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p < 0.05 compared to controls and was calculated using GraphPad Prism.

Article Snippet: The plasmid (CD63-GFP) (Origene cat# RG201733) was used to generate the stable C4-2B-CD63-GFP cells.

Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay