Structured Review

Abcam anti cd81
Polymersomes superselectivity. Schematics of the binding between Psomes of radius and SRB1, CD36, and <t>CD81</t> receptors (a). The fraction of bound Psomes (blue) and the binding energy to FaDu cells per Psome (orange) as a function of the PMPC degree of polymerization, N PC . Note the experimental values were measured from the cellular uptake at 2 h (b).
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1) Product Images from "A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting"

Article Title: A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting

Journal: ACS Central Science

doi: 10.1021/acscentsci.2c00146

Polymersomes superselectivity. Schematics of the binding between Psomes of radius and SRB1, CD36, and CD81 receptors (a). The fraction of bound Psomes (blue) and the binding energy to FaDu cells per Psome (orange) as a function of the PMPC degree of polymerization, N PC . Note the experimental values were measured from the cellular uptake at 2 h (b).
Figure Legend Snippet: Polymersomes superselectivity. Schematics of the binding between Psomes of radius and SRB1, CD36, and CD81 receptors (a). The fraction of bound Psomes (blue) and the binding energy to FaDu cells per Psome (orange) as a function of the PMPC degree of polymerization, N PC . Note the experimental values were measured from the cellular uptake at 2 h (b).

Techniques Used: Binding Assay

PMPC free chain binding to SRB1, CD36, and CD81 receptors. Autodock vina estimated relative binding affinities are shown as swarm plots at increasing number of PMPC chains ( N PC ) for SRB1, CD36, and CD81 (a). All-atom depiction of the PMPC free chain (for N PC with highest affinity) binding site, shown as VDW surface in magenta. For CD36 and SRB1, glycans are shown as licorice and their VDW surface is also overlay. The secondary structure of the three receptors is shown as a cartoon in silver. Cellular membrane is indicated in gray. In the CD81 case, two binding modes are depicted (b). PMPC free chain binding site inside CD36 and SRB1 (c). PMPC free chain docking pose versus MD stable pose onto CD81 (d).
Figure Legend Snippet: PMPC free chain binding to SRB1, CD36, and CD81 receptors. Autodock vina estimated relative binding affinities are shown as swarm plots at increasing number of PMPC chains ( N PC ) for SRB1, CD36, and CD81 (a). All-atom depiction of the PMPC free chain (for N PC with highest affinity) binding site, shown as VDW surface in magenta. For CD36 and SRB1, glycans are shown as licorice and their VDW surface is also overlay. The secondary structure of the three receptors is shown as a cartoon in silver. Cellular membrane is indicated in gray. In the CD81 case, two binding modes are depicted (b). PMPC free chain binding site inside CD36 and SRB1 (c). PMPC free chain docking pose versus MD stable pose onto CD81 (d).

Techniques Used: Binding Assay

PMPC binding induces CD81 opening and membrane curvature. Conformational landscape explored by the PMPC free chain ( N PC = 4) on the surface of CD81. The free-energy landscape is projected onto the distance of the center of mass of the PMPC free chain and the surface of CD81 and the polar angle (described in Figure 2 d) (a). Time evolution of the angles describing the allosteric movement induced by PMPC onto the helix TM4 of CD81, colored according to simulation time. The TM4-EC2 angle is highlighted in red and the TM4 kink in lavender (b). Initial membrane curvature represented by the phosphorus atoms of the lipid heads (c). All-atom depiction of CD81 (as white cartoon) and the PMPC chain (magenta surface) along the MD simulation. The TM4 is colored according to simulation time. The angles describing the allosteric movement are indicated in snapshot 1 (TM4-EC2) and 7 (TM4-kink) (d). Final membrane curvature (e).
Figure Legend Snippet: PMPC binding induces CD81 opening and membrane curvature. Conformational landscape explored by the PMPC free chain ( N PC = 4) on the surface of CD81. The free-energy landscape is projected onto the distance of the center of mass of the PMPC free chain and the surface of CD81 and the polar angle (described in Figure 2 d) (a). Time evolution of the angles describing the allosteric movement induced by PMPC onto the helix TM4 of CD81, colored according to simulation time. The TM4-EC2 angle is highlighted in red and the TM4 kink in lavender (b). Initial membrane curvature represented by the phosphorus atoms of the lipid heads (c). All-atom depiction of CD81 (as white cartoon) and the PMPC chain (magenta surface) along the MD simulation. The TM4 is colored according to simulation time. The angles describing the allosteric movement are indicated in snapshot 1 (TM4-EC2) and 7 (TM4-kink) (d). Final membrane curvature (e).

Techniques Used: Binding Assay

Cellular uptake of PMPC polymersomes. Expression levels of CD36, SRB1, and CD81 in FaDu, HDF, and THP-1 cells were assessed by Western blot relative to GAPDH used as a loading control (a). Immunofluorescence micrographs of all three cell lines with CD36, SRB1, and CD81 labeled (b). Fluorescent-labeled PMPC Psome uptake in FaDu, HDF, and THP-1 cells as a function of time measured by flow cytometry (c). Cytofluorimetry-based quantification of PMPC 25 –PDPA 70 Psome uptake in FaDu, HDF, and THP-1 cells upon treatment with the specific blocking antibodies against SRBI, CD36, and CD81. Experimental error is represented as shaded error bands. (d). PLA quantification was relative to untreated FaDu cells showing the clustering of SRB1, CD36, and CD81 receptors following 1 h incubation with 0.1 mg/mL PMPC 25 –PDPA 70 Psomes (**** P
Figure Legend Snippet: Cellular uptake of PMPC polymersomes. Expression levels of CD36, SRB1, and CD81 in FaDu, HDF, and THP-1 cells were assessed by Western blot relative to GAPDH used as a loading control (a). Immunofluorescence micrographs of all three cell lines with CD36, SRB1, and CD81 labeled (b). Fluorescent-labeled PMPC Psome uptake in FaDu, HDF, and THP-1 cells as a function of time measured by flow cytometry (c). Cytofluorimetry-based quantification of PMPC 25 –PDPA 70 Psome uptake in FaDu, HDF, and THP-1 cells upon treatment with the specific blocking antibodies against SRBI, CD36, and CD81. Experimental error is represented as shaded error bands. (d). PLA quantification was relative to untreated FaDu cells showing the clustering of SRB1, CD36, and CD81 receptors following 1 h incubation with 0.1 mg/mL PMPC 25 –PDPA 70 Psomes (**** P

Techniques Used: Expressing, Western Blot, Immunofluorescence, Labeling, Flow Cytometry, Blocking Assay, Proximity Ligation Assay, Incubation

2) Product Images from "Regenerative and protective effects of dMSC-sEVs on high-glucose-induced senescent fibroblasts by suppressing RAGE pathway and activating Smad pathway"

Article Title: Regenerative and protective effects of dMSC-sEVs on high-glucose-induced senescent fibroblasts by suppressing RAGE pathway and activating Smad pathway

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-020-01681-z

Characterization of dMSC-sEVs. a Ultrastructure of dMSC-sEVs, scale bar = 50 nm. b Dynamic tracking capture and particle size distribution were measured by nanoparticle tracking analyzer. c The expression level of CD9, CD63, CD81, TSG101, and Grp94. d Internalization assay of dMSCs-sEVs by HDFs. dMSC-sEVs were marked by the green fluorescence (PKH-67) and cytoskeleton were marked by red fluorescence (phalloidin). Scale bar = 25 μm
Figure Legend Snippet: Characterization of dMSC-sEVs. a Ultrastructure of dMSC-sEVs, scale bar = 50 nm. b Dynamic tracking capture and particle size distribution were measured by nanoparticle tracking analyzer. c The expression level of CD9, CD63, CD81, TSG101, and Grp94. d Internalization assay of dMSCs-sEVs by HDFs. dMSC-sEVs were marked by the green fluorescence (PKH-67) and cytoskeleton were marked by red fluorescence (phalloidin). Scale bar = 25 μm

Techniques Used: Expressing, Fluorescence

3) Product Images from "Exosomes derived from cardiac telocytes exert positive effects on endothelial cells"

Article Title: Exosomes derived from cardiac telocytes exert positive effects on endothelial cells

Journal: American Journal of Translational Research

doi:

Isolation of exosomes from telocytes (TCs) and investigation of their uptake by endothelial cells (ECs). A. Representative electron microscopy image of TC exosomes. The diameter of this exosome was measured to be 105.59 nm. Scale bar: 50 nm. B, C. To confirm their identity, exosomal preparations were compared with cells lysates by Western blotting. The blot shown an enrichment of the exosomal markers Alix, CD9, CD63, HSP90, CD81 and TSG101 but the absence of calnexin, an endoplasmic reticulum protein. β-actin was used as a loading control. D. Exosome identity was further confirmed with a qNano analysis, which measured the diameters of the particles with respect to concentration. Most particles ranged from 50 to 150 nm in diameter. E. Isolated exosomes from TCs were labeled with PKH26 (red) and added to the culture medium of GFP-labeled ECs (green). After 12 h, cells were stained with DAPI (blue) to label cell nuclei and confocal imaging showed that the exosomes were present in the cytoplasm of ECs. Scale bar: 10 µm. F. TCs were transfected with cel-miR-39 or left un-transfected (control). Cultured ECs were then treated with exosomes isolated from control or cel-miR-39-transfected TCs for the indicated times. The RNA levels of cel-miR-39 were then measured in ECs using RT-PCR. ****, P
Figure Legend Snippet: Isolation of exosomes from telocytes (TCs) and investigation of their uptake by endothelial cells (ECs). A. Representative electron microscopy image of TC exosomes. The diameter of this exosome was measured to be 105.59 nm. Scale bar: 50 nm. B, C. To confirm their identity, exosomal preparations were compared with cells lysates by Western blotting. The blot shown an enrichment of the exosomal markers Alix, CD9, CD63, HSP90, CD81 and TSG101 but the absence of calnexin, an endoplasmic reticulum protein. β-actin was used as a loading control. D. Exosome identity was further confirmed with a qNano analysis, which measured the diameters of the particles with respect to concentration. Most particles ranged from 50 to 150 nm in diameter. E. Isolated exosomes from TCs were labeled with PKH26 (red) and added to the culture medium of GFP-labeled ECs (green). After 12 h, cells were stained with DAPI (blue) to label cell nuclei and confocal imaging showed that the exosomes were present in the cytoplasm of ECs. Scale bar: 10 µm. F. TCs were transfected with cel-miR-39 or left un-transfected (control). Cultured ECs were then treated with exosomes isolated from control or cel-miR-39-transfected TCs for the indicated times. The RNA levels of cel-miR-39 were then measured in ECs using RT-PCR. ****, P

Techniques Used: Isolation, Electron Microscopy, Western Blot, Concentration Assay, Labeling, Staining, Imaging, Transfection, Cell Culture, Reverse Transcription Polymerase Chain Reaction

4) Product Images from "Exosomal transfer of miR‐106a‐5p contributes to cisplatin resistance and tumorigenesis in nasopharyngeal carcinoma, et al. Exosomal transfer of miR‐106a‐5p contributes to cisplatin resistance and tumorigenesis in nasopharyngeal carcinoma"

Article Title: Exosomal transfer of miR‐106a‐5p contributes to cisplatin resistance and tumorigenesis in nasopharyngeal carcinoma, et al. Exosomal transfer of miR‐106a‐5p contributes to cisplatin resistance and tumorigenesis in nasopharyngeal carcinoma

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.16801

miR‐106a‐5p is enriched along with an increased number of exosomes in cisplatin‐resistant CNE1 cells. (A) Plate colony formation assays were used to estimate the cisplatin resistance of CNE1r cells. (B) Relative cell viability of CNE1s and CNE1r cells with cisplatin treatment(0, 1, 2, 4, 8, 16 and 32 μg/ml) for 48 h. (C) Relative expression of miR‐106a‐5p in CNE1s and CNE1r was measured by RT‐qPCR. (D) Transmission electron microscopic images of exosomes derived from CNE1s and CNE1r. (E) Exosomal‐positive markers CD81 and HSP70 were detected in CNE1s‐derived and CNE1r‐derived exosomes using Western blotting. (F) Flow cytometry of the exosomal surface marker CD81 and HSP70 level. (G) The size distribution of the isolated exosomes was measured by high‐sensitivity flow cytometry. CNE1s, cisplatin‐sensitive CNE1 cells; CNE1r, cisplatin‐resistant CNE1 cells. In all experiments, bars represent mean ± SD for three replicates. * p
Figure Legend Snippet: miR‐106a‐5p is enriched along with an increased number of exosomes in cisplatin‐resistant CNE1 cells. (A) Plate colony formation assays were used to estimate the cisplatin resistance of CNE1r cells. (B) Relative cell viability of CNE1s and CNE1r cells with cisplatin treatment(0, 1, 2, 4, 8, 16 and 32 μg/ml) for 48 h. (C) Relative expression of miR‐106a‐5p in CNE1s and CNE1r was measured by RT‐qPCR. (D) Transmission electron microscopic images of exosomes derived from CNE1s and CNE1r. (E) Exosomal‐positive markers CD81 and HSP70 were detected in CNE1s‐derived and CNE1r‐derived exosomes using Western blotting. (F) Flow cytometry of the exosomal surface marker CD81 and HSP70 level. (G) The size distribution of the isolated exosomes was measured by high‐sensitivity flow cytometry. CNE1s, cisplatin‐sensitive CNE1 cells; CNE1r, cisplatin‐resistant CNE1 cells. In all experiments, bars represent mean ± SD for three replicates. * p

Techniques Used: Expressing, Quantitative RT-PCR, Transmission Assay, Derivative Assay, Western Blot, Flow Cytometry, Marker, Isolation

5) Product Images from "The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication"

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

Journal: Nature Communications

doi: 10.1038/s41467-019-08299-7

Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles
Figure Legend Snippet: Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

Techniques Used: Expressing, Over Expression

HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)
Figure Legend Snippet: HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

REV-ERB agonists inhibit hepatitis C virus (HCV) entry. a REV-ERB agonists inhibit BMAL1 transcription. Huh-7 cells were treated with REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and Bmal1 mRNA levels quantified by qRT-PCR, respectively (mean ± S.E.M., n = 7, Kruskal–Wallis ANOVA with Dunn’s test). b , c REV-ERB agonists modulate HCV receptor expression. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and the cells lysed, total protein measured and assessed for CD81, claudin-1 and occludin expression together with the housekeeping GAPDH by western blotting or mass spectrometric analysis (mean ± S.E.M., n = 3, Mann–Whitney test). d REV-ERB agonists inhibit HCV entry. Huh-7 cells were treated with increasing dose of REV-ERB agonists SR9009 or GSK2667 for 24 h, inoculated with HCVpp and infection assessed 24 h later (mean ± S.E.M., n = 5). e REV-ERB agonists inhibit HCVpp bearing patient-derived glycoproteins. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h, infected with HCVpp bearing patient-derived envelope glycoproteins and infection assessed 24 h later. In all cases, data are expressed relative to untreated (Ctrl) cells. (Mean ± S.E.M., n = 3, Kruskal–Wallis ANOVA)
Figure Legend Snippet: REV-ERB agonists inhibit hepatitis C virus (HCV) entry. a REV-ERB agonists inhibit BMAL1 transcription. Huh-7 cells were treated with REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and Bmal1 mRNA levels quantified by qRT-PCR, respectively (mean ± S.E.M., n = 7, Kruskal–Wallis ANOVA with Dunn’s test). b , c REV-ERB agonists modulate HCV receptor expression. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and the cells lysed, total protein measured and assessed for CD81, claudin-1 and occludin expression together with the housekeeping GAPDH by western blotting or mass spectrometric analysis (mean ± S.E.M., n = 3, Mann–Whitney test). d REV-ERB agonists inhibit HCV entry. Huh-7 cells were treated with increasing dose of REV-ERB agonists SR9009 or GSK2667 for 24 h, inoculated with HCVpp and infection assessed 24 h later (mean ± S.E.M., n = 5). e REV-ERB agonists inhibit HCVpp bearing patient-derived glycoproteins. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h, infected with HCVpp bearing patient-derived envelope glycoproteins and infection assessed 24 h later. In all cases, data are expressed relative to untreated (Ctrl) cells. (Mean ± S.E.M., n = 3, Kruskal–Wallis ANOVA)

Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY, Infection, Derivative Assay

REV-ERBα inhibits hepatitis C virus (HCV) RNA replication. a Silencing Rev-erbα increases HCV replication. Huh-7 cells supporting a HCV JFH-1-LUC replicon were transduced with lentivirus encoding sh Rev-erbα or control and silencing confirmed by measuring Rev-erbα mRNA and protein expression levels (mean ± S.E.M., n = 4, Mann–Whitney test). Densitometric analysis quantified REV-ERB in individual samples and was normalised to its own GAPDH loading control. HCV replication-dependent reporter activity was measured and expressed relative to control (shCtrl) cells (mean ± S.E.M., n = 6, Mann–Whitney test). b Anti-viral activity of SR9009 agonist is dependent on REV-ERB expression levels. sh Rev-eRbα and Ctrl HCV JFH-1 replicon cells described in ( a ) were treated with REV-ERB agonist SR9009 for 24 h, viral replication measured and the concentration of agonist required to inhibit viral replication by 50% defined (IC 50 ) (mean ± S.E.M., n = 3). c REV-ERBα overexpression inhibits HCV RNA replication. Huh-7 cells stably supporting a HCV JFH-1-LUC replicon were transfected with empty plasmid or vector expressing REV-ERBα and 48 h later protein expression assessed by western blotting and viral replication measured (mean ± S.E.M., n = 4, Mann–Whitney statistical test). Data are plotted relative to Ctrl untreated cells. d REV-ERB agonists cure HCV-infected cells. HCVcc SA13/JFH-1 infected Huh-7 cells were treated with increasing concentrations of REV-ERB agonists for 24 h and viral RNA or NS5A-expressing cells quantified and data expressed relative to Ctrl untreated cells. The experiment was performed in the presence of a neutralising anti-CD81 antibody to limit secondary rounds of infection (mean ± S.E.M., n = 3). e REV-ERB ligands inhibit the replication of diverse HCV genotypes. Huh-7 cells transiently supporting HCV sub-genomic replicons representing genotypes 1–3 were treated with the REV-ERB agonists SR9009 or GSK2667 and replication assessed 24 h later. The dose of agonist required to inhibit HCV RNA replication by 50% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)
Figure Legend Snippet: REV-ERBα inhibits hepatitis C virus (HCV) RNA replication. a Silencing Rev-erbα increases HCV replication. Huh-7 cells supporting a HCV JFH-1-LUC replicon were transduced with lentivirus encoding sh Rev-erbα or control and silencing confirmed by measuring Rev-erbα mRNA and protein expression levels (mean ± S.E.M., n = 4, Mann–Whitney test). Densitometric analysis quantified REV-ERB in individual samples and was normalised to its own GAPDH loading control. HCV replication-dependent reporter activity was measured and expressed relative to control (shCtrl) cells (mean ± S.E.M., n = 6, Mann–Whitney test). b Anti-viral activity of SR9009 agonist is dependent on REV-ERB expression levels. sh Rev-eRbα and Ctrl HCV JFH-1 replicon cells described in ( a ) were treated with REV-ERB agonist SR9009 for 24 h, viral replication measured and the concentration of agonist required to inhibit viral replication by 50% defined (IC 50 ) (mean ± S.E.M., n = 3). c REV-ERBα overexpression inhibits HCV RNA replication. Huh-7 cells stably supporting a HCV JFH-1-LUC replicon were transfected with empty plasmid or vector expressing REV-ERBα and 48 h later protein expression assessed by western blotting and viral replication measured (mean ± S.E.M., n = 4, Mann–Whitney statistical test). Data are plotted relative to Ctrl untreated cells. d REV-ERB agonists cure HCV-infected cells. HCVcc SA13/JFH-1 infected Huh-7 cells were treated with increasing concentrations of REV-ERB agonists for 24 h and viral RNA or NS5A-expressing cells quantified and data expressed relative to Ctrl untreated cells. The experiment was performed in the presence of a neutralising anti-CD81 antibody to limit secondary rounds of infection (mean ± S.E.M., n = 3). e REV-ERB ligands inhibit the replication of diverse HCV genotypes. Huh-7 cells transiently supporting HCV sub-genomic replicons representing genotypes 1–3 were treated with the REV-ERB agonists SR9009 or GSK2667 and replication assessed 24 h later. The dose of agonist required to inhibit HCV RNA replication by 50% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

Techniques Used: Transduction, Expressing, MANN-WHITNEY, Activity Assay, Concentration Assay, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Infection

6) Product Images from "Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects"

Article Title: Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects

Journal: Signal Transduction and Targeted Therapy

doi: 10.1038/s41392-022-00894-3

Characterizations of M1EVs based formulations and evaluations of the penetration capacity and synergistic anti-tumor efficacy in vitro. a . TEM image of M1EVs. Scale bar: 100 nm. b . ProteinSimple ® capillary immunoassay (Wes) analysis of CD9, CD81, ALIX, TSG101, iNOS, F4/80, and GAPDH in M1 macrophages and M1EVs. c . Confocal laser scanning microscopy (CLSM) images of AQ4N-M1EVs (Top, green: M1EVs; red: AQ4N) and Ce6-M1EVs (bottom, green: M1EVs; red: Ce6). All images have the same scale of 1 μm. d . Representative flow cytometry analysis images of M1EVs (top) and TA-M1EVs (M1EVs containing AQ4N and TRMRA in place of Ce6 due to the overlayed spectrum with AQ4N) (bottom). e . Production of ROS with Ce6, CPPO/Ce6, CC-M1EVs, and CCA-M1EVs in buffers with different H 2 O 2 concentrations, where A 0 and A were the absorbance of ABDA at 399 nm before and after H 2 O 2 addition ( n = 3). f . Cumulative AQ4N release profiles of CCA-M1EVs before and after H 2 O 2 treatment in PBS buffer ( n = 3). g . Consumption of oxygen with different formulations after H 2 O 2 treatment in PBS buffer ( n = 3). h . Quantification of the AQ4/AQ4N ratio after different treatments based on high-performance liquid chromatography (HPLC) analysis. i . Illustration of in vitro BBB and TME model. The Transwell TM co-culture system containing bEnd.3 cells in the upper chamber and a combination of U87MG glioma cells and macrophages in the bottom chamber under hypoxic condition. j . CLSM images of bEnd.3 cells with different treatments. Scale bar: 5 μm. (green: ZO-1, red: EVs). k . Accumulative penetration efficiency of M1EVs, CC-M1EVs, A-M1EVs, and CCA-M1EVs labeled with DiD through a monolayer bEnd.3 layer at different time points ( n = 3). l . Flow cytometry analysis of the M2/M1 ratio in the lower chamber after incubation with different EV designs ( n = 3). m . Production of H 2 O 2 with different treatments in the lower chamber (Amplex Red Hydrogen Peroxide Assay Kit) ( n = 3). n . Assessment of intracellular ROS (labeled by DCFH-DA) of U87MG cells in the lower chamber ( n = 3). o . Flow cytometry analysis of the cell-death-inducing effect of different formulations on U87MG cells in the lower chamber (Annexin V and PI in the dead cell apoptosis kit) ( n = 3). Statistical significance was calculated via one-way ANOVA with a Kruskal-Wallis test ( e , g , l , m , n , and o ) or unpaired two-tailed Student’s t -test ( f ). ns, not significant
Figure Legend Snippet: Characterizations of M1EVs based formulations and evaluations of the penetration capacity and synergistic anti-tumor efficacy in vitro. a . TEM image of M1EVs. Scale bar: 100 nm. b . ProteinSimple ® capillary immunoassay (Wes) analysis of CD9, CD81, ALIX, TSG101, iNOS, F4/80, and GAPDH in M1 macrophages and M1EVs. c . Confocal laser scanning microscopy (CLSM) images of AQ4N-M1EVs (Top, green: M1EVs; red: AQ4N) and Ce6-M1EVs (bottom, green: M1EVs; red: Ce6). All images have the same scale of 1 μm. d . Representative flow cytometry analysis images of M1EVs (top) and TA-M1EVs (M1EVs containing AQ4N and TRMRA in place of Ce6 due to the overlayed spectrum with AQ4N) (bottom). e . Production of ROS with Ce6, CPPO/Ce6, CC-M1EVs, and CCA-M1EVs in buffers with different H 2 O 2 concentrations, where A 0 and A were the absorbance of ABDA at 399 nm before and after H 2 O 2 addition ( n = 3). f . Cumulative AQ4N release profiles of CCA-M1EVs before and after H 2 O 2 treatment in PBS buffer ( n = 3). g . Consumption of oxygen with different formulations after H 2 O 2 treatment in PBS buffer ( n = 3). h . Quantification of the AQ4/AQ4N ratio after different treatments based on high-performance liquid chromatography (HPLC) analysis. i . Illustration of in vitro BBB and TME model. The Transwell TM co-culture system containing bEnd.3 cells in the upper chamber and a combination of U87MG glioma cells and macrophages in the bottom chamber under hypoxic condition. j . CLSM images of bEnd.3 cells with different treatments. Scale bar: 5 μm. (green: ZO-1, red: EVs). k . Accumulative penetration efficiency of M1EVs, CC-M1EVs, A-M1EVs, and CCA-M1EVs labeled with DiD through a monolayer bEnd.3 layer at different time points ( n = 3). l . Flow cytometry analysis of the M2/M1 ratio in the lower chamber after incubation with different EV designs ( n = 3). m . Production of H 2 O 2 with different treatments in the lower chamber (Amplex Red Hydrogen Peroxide Assay Kit) ( n = 3). n . Assessment of intracellular ROS (labeled by DCFH-DA) of U87MG cells in the lower chamber ( n = 3). o . Flow cytometry analysis of the cell-death-inducing effect of different formulations on U87MG cells in the lower chamber (Annexin V and PI in the dead cell apoptosis kit) ( n = 3). Statistical significance was calculated via one-way ANOVA with a Kruskal-Wallis test ( e , g , l , m , n , and o ) or unpaired two-tailed Student’s t -test ( f ). ns, not significant

Techniques Used: In Vitro, Transmission Electron Microscopy, Confocal Laser Scanning Microscopy, Flow Cytometry, High Performance Liquid Chromatography, Co-Culture Assay, Labeling, Incubation, Amplex Red Hydrogen Peroxide Assay, Two Tailed Test

7) Product Images from "Exosomes secreted by human urine-derived stem cells could prevent kidney complications from type I diabetes in rats"

Article Title: Exosomes secreted by human urine-derived stem cells could prevent kidney complications from type I diabetes in rats

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-016-0287-2

Characteristics of USCs and USCs-Exo. a ) The morphology and growth of USCs. Scale bar = 200 μm. b ) USCs were characterized by flow cytometry using the surface markers CD29, CD90, CD73, CD44, CD34, CD45 and HLA-DR. White solid peaks represent the isotype controls and the grey solid peak represents the marker indicated. c ) Morphology of USCs-Exo under a transmission electron microscopy. d ) TRPS measurement showed that the size range of USCs-Exo concentrated at 50–100 nm, and the measured mean concentration (particles/ml) of USCs-Exo was 5.2E + 009. e ) Western blotting analysis of exosome-specific CD9, CD63 and CD81 proteins in USCs and USCs-Exo. USC Urine-derived stem cell, USCs-Exo Exosome from urine-derived stem cells
Figure Legend Snippet: Characteristics of USCs and USCs-Exo. a ) The morphology and growth of USCs. Scale bar = 200 μm. b ) USCs were characterized by flow cytometry using the surface markers CD29, CD90, CD73, CD44, CD34, CD45 and HLA-DR. White solid peaks represent the isotype controls and the grey solid peak represents the marker indicated. c ) Morphology of USCs-Exo under a transmission electron microscopy. d ) TRPS measurement showed that the size range of USCs-Exo concentrated at 50–100 nm, and the measured mean concentration (particles/ml) of USCs-Exo was 5.2E + 009. e ) Western blotting analysis of exosome-specific CD9, CD63 and CD81 proteins in USCs and USCs-Exo. USC Urine-derived stem cell, USCs-Exo Exosome from urine-derived stem cells

Techniques Used: Flow Cytometry, Cytometry, Marker, Transmission Assay, Electron Microscopy, Concentration Assay, Western Blot, Derivative Assay

8) Product Images from "Exosomes derived from cardiomyocytes promote cardiac fibrosis via myocyte-fibroblast cross-talk"

Article Title: Exosomes derived from cardiomyocytes promote cardiac fibrosis via myocyte-fibroblast cross-talk

Journal: American Journal of Translational Research

doi:

Hypoxia-stimulated or Ang II-induced cardiomyocytes secrete exosomes that are taken up by cardiac fibroblasts. A. Electron micrographs of exosomes. The diameter of an exosome is 106 nm. Scale bar: 50 nm. B. Western blotting showed the exosome markers CD9, CD63, and CD81 and the endoplasmic reticulum marker calnexin. C. qNano analysis of exosomes showed the particle diameter and concentration. The diameter ranged from about 50 nm to 150 nm. Blue, exosomes from PBS-treated cardiomyocytes; green, exosomes from hypoxic cardiomyocytes; red, exosomes from Ang II-induced cardiomyocytes. D. cel-miR-39-transfected cardiomyocytes. Fibroblasts were then exposed to exosomes extracted from cel-miR-39-transfected cardiomyocytes or untransfected (control) for the stated times. *P
Figure Legend Snippet: Hypoxia-stimulated or Ang II-induced cardiomyocytes secrete exosomes that are taken up by cardiac fibroblasts. A. Electron micrographs of exosomes. The diameter of an exosome is 106 nm. Scale bar: 50 nm. B. Western blotting showed the exosome markers CD9, CD63, and CD81 and the endoplasmic reticulum marker calnexin. C. qNano analysis of exosomes showed the particle diameter and concentration. The diameter ranged from about 50 nm to 150 nm. Blue, exosomes from PBS-treated cardiomyocytes; green, exosomes from hypoxic cardiomyocytes; red, exosomes from Ang II-induced cardiomyocytes. D. cel-miR-39-transfected cardiomyocytes. Fibroblasts were then exposed to exosomes extracted from cel-miR-39-transfected cardiomyocytes or untransfected (control) for the stated times. *P

Techniques Used: Western Blot, Marker, Concentration Assay, Transfection

9) Product Images from "Extracellular vesicles derived from human dental pulp stem cells promote osteogenesis of adipose-derived stem cells via the MAPK pathway"

Article Title: Extracellular vesicles derived from human dental pulp stem cells promote osteogenesis of adipose-derived stem cells via the MAPK pathway

Journal: Journal of Tissue Engineering

doi: 10.1177/2041731420975569

Characteristics of hDPSC-EVs. (a) TEM observation of hDPSC-EVs; (b) particle size identification of hDPSC-EVs detected by NTA; (c) western blotting analysis of CD9, CD63, CD81, and TSG101 in EVs.
Figure Legend Snippet: Characteristics of hDPSC-EVs. (a) TEM observation of hDPSC-EVs; (b) particle size identification of hDPSC-EVs detected by NTA; (c) western blotting analysis of CD9, CD63, CD81, and TSG101 in EVs.

Techniques Used: Transmission Electron Microscopy, Western Blot

10) Product Images from "In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53"

Article Title: In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200609050

The intracellular VCCs are accessible from the cell surface. (A–J) The fluid tracer HRP has access to the VCCs. MDMs infected with HIV for 8 d (A–E) or uninfected MDMs cultured for 13 d and incubated with anti-CD81 for 3 h at 37°C (F-J) were cooled on ice and incubated for 1 h at 4°C with 10 mg/ml HRP. Cells were fixed and processed for cryosectioning. Semithin (0.5 μm thick) sections were triple-stained with anti-p17 mouse IgG2a (A and F, visualized with anti–IgG2a-Alexa Fluor 488, green), anti-CD81 mouse IgG1 (B, anti-IgG1-Alexa Fluor 594, red, or in G, second antibody only) and rabbit anti-HRP (C and H, anti–rabbit-Alexa Fluor 697, blue). Merged immunofluorescence (D and I) and phase-contrast (E and J) images of the sections. HRP reached the CD81-labeled virus-containing structures (D) or the structure containing the fed anti-CD81 (I). (K–Q) MDMs infected with HIV for 13 d were fixed in the presence of RR and embedded in Epon. RR marks surface-accessible membranes with a flocculated, electron-dense deposit. In K, virus particles inside VCCs were coated with RR. L shows extended closely apposed membrane sheets (black arrowheads) near the cell surface (Su) or deep in the cell. The asterisks mark RR-stained membranes cut parallel to the section; the area under the white asterisk links these structures to the cell surface (see the enlargement in M). #, interconnected spongelike membranes deep in the cell. V, virus particles; Li, lipid droplets. (N–Q) Sections 28, 27, 26, and 24, respectively, of a set of 41 serial sections. The black arrowheads in (N and O) mark a thin channel of RR-stained membranes from the cell surface (Su) into VCC 1. In P, VCCs 1 and 2 are coalescing (the edge of the compartment membrane is cut grazingly at the white arrowhead). In Q, VCCs 1 and 2 are fused, and are connected to VCC 3 through a narrow gap (arrow). Bars: (D and I) 10 μm; (K) 200 nm; (L and Q) 500 nm.
Figure Legend Snippet: The intracellular VCCs are accessible from the cell surface. (A–J) The fluid tracer HRP has access to the VCCs. MDMs infected with HIV for 8 d (A–E) or uninfected MDMs cultured for 13 d and incubated with anti-CD81 for 3 h at 37°C (F-J) were cooled on ice and incubated for 1 h at 4°C with 10 mg/ml HRP. Cells were fixed and processed for cryosectioning. Semithin (0.5 μm thick) sections were triple-stained with anti-p17 mouse IgG2a (A and F, visualized with anti–IgG2a-Alexa Fluor 488, green), anti-CD81 mouse IgG1 (B, anti-IgG1-Alexa Fluor 594, red, or in G, second antibody only) and rabbit anti-HRP (C and H, anti–rabbit-Alexa Fluor 697, blue). Merged immunofluorescence (D and I) and phase-contrast (E and J) images of the sections. HRP reached the CD81-labeled virus-containing structures (D) or the structure containing the fed anti-CD81 (I). (K–Q) MDMs infected with HIV for 13 d were fixed in the presence of RR and embedded in Epon. RR marks surface-accessible membranes with a flocculated, electron-dense deposit. In K, virus particles inside VCCs were coated with RR. L shows extended closely apposed membrane sheets (black arrowheads) near the cell surface (Su) or deep in the cell. The asterisks mark RR-stained membranes cut parallel to the section; the area under the white asterisk links these structures to the cell surface (see the enlargement in M). #, interconnected spongelike membranes deep in the cell. V, virus particles; Li, lipid droplets. (N–Q) Sections 28, 27, 26, and 24, respectively, of a set of 41 serial sections. The black arrowheads in (N and O) mark a thin channel of RR-stained membranes from the cell surface (Su) into VCC 1. In P, VCCs 1 and 2 are coalescing (the edge of the compartment membrane is cut grazingly at the white arrowhead). In Q, VCCs 1 and 2 are fused, and are connected to VCC 3 through a narrow gap (arrow). Bars: (D and I) 10 μm; (K) 200 nm; (L and Q) 500 nm.

Techniques Used: Infection, Cell Culture, Incubation, Staining, Immunofluorescence, Labeling

Distribution of tetraspanins in uninfected MDMs. MDMs were grown for 16 d, fixed, and stained with mAbs against CD63 (A), CD9 (B), or CD53 (C) and anti–mouse Alexa Fluor 488. CD63 labeled a network of tubular structures, whereas CD9 and CD53 localized to the cell surface and discrete intracellular puncta. To identify these puncta, cells were double stained with mouse IgG1 mAbs to LAMP-1, CD81, CD53, or EEA1, detected with anti–mouse IgG1-Alexa Fluor 594 (red), and IgG2b mAbs to CD63 or CD9, detected with anti–mouse IgG2b-Alexa Fluor 488 (green). CD63 staining overlapped with LAMP-1 (D), but there was almost no colocalization of CD63 with CD81 (E). CD9 labeling showed nearly complete overlap with CD53 (F) or CD81 (G). Neither CD63 (H) nor CD9 (I) colocalized with EEA1. The images show single confocal sections ∼1 μm from the bottom of the cell. For D–G and I, the insets show enlargements. Bars, 10 μm.
Figure Legend Snippet: Distribution of tetraspanins in uninfected MDMs. MDMs were grown for 16 d, fixed, and stained with mAbs against CD63 (A), CD9 (B), or CD53 (C) and anti–mouse Alexa Fluor 488. CD63 labeled a network of tubular structures, whereas CD9 and CD53 localized to the cell surface and discrete intracellular puncta. To identify these puncta, cells were double stained with mouse IgG1 mAbs to LAMP-1, CD81, CD53, or EEA1, detected with anti–mouse IgG1-Alexa Fluor 594 (red), and IgG2b mAbs to CD63 or CD9, detected with anti–mouse IgG2b-Alexa Fluor 488 (green). CD63 staining overlapped with LAMP-1 (D), but there was almost no colocalization of CD63 with CD81 (E). CD9 labeling showed nearly complete overlap with CD53 (F) or CD81 (G). Neither CD63 (H) nor CD9 (I) colocalized with EEA1. The images show single confocal sections ∼1 μm from the bottom of the cell. For D–G and I, the insets show enlargements. Bars, 10 μm.

Techniques Used: Staining, Labeling

CD9 and CD81 in VCCs in MDMs. Ultrathin cryosections from MDMs infected with HIV for 7 d were double labeled with mouse mAbs against CD9 (A–C) or CD81 (D and E) and 15 nm PAG, followed by rabbit anti-p17 and 5 nm PAG. HIV virions are identified by their characteristic size (120–140 nm diam) and morphology and by labeling with 5 nm PAG (some virions are marked V in A–E). CD9 and CD81 are found in the VCCs, occasionally even in the viral envelopes, as well as over internal membranes of various sizes and including small (50 nm diam) vesicles (A and D, black arrows). Note the thick protein coat on the apposed membrane sheets in C (white arrowheads) and the extended areas of closely apposed membrane sheets in E (black arrowheads). Bars, 200 nm.
Figure Legend Snippet: CD9 and CD81 in VCCs in MDMs. Ultrathin cryosections from MDMs infected with HIV for 7 d were double labeled with mouse mAbs against CD9 (A–C) or CD81 (D and E) and 15 nm PAG, followed by rabbit anti-p17 and 5 nm PAG. HIV virions are identified by their characteristic size (120–140 nm diam) and morphology and by labeling with 5 nm PAG (some virions are marked V in A–E). CD9 and CD81 are found in the VCCs, occasionally even in the viral envelopes, as well as over internal membranes of various sizes and including small (50 nm diam) vesicles (A and D, black arrows). Note the thick protein coat on the apposed membrane sheets in C (white arrowheads) and the extended areas of closely apposed membrane sheets in E (black arrowheads). Bars, 200 nm.

Techniques Used: Infection, Labeling

Uptake of antibodies directed against tetraspanins. MDMs cultured for 13 d were incubated for 3 h at 37°C in medium containing mAbs against CD81, CD53, or CD9 (fed). Cells were then fixed, permeabilized, and stained with antibodies against a different tetraspanin (steady-state). Antibodies were detected with isotype-specific secondary antibodies (red, anti–mouse IgG1-Alexa Fluor 594, for CD81 and CD53; green, anti–mouse IgG2b-Alexa Fluor 488, for CD9). CD9 colocalized with internalized anti-CD81 (A) or anti-CD53 (B). Similarly, internalized anti-CD9 was found in intracellular structures positive for CD53 (C) or CD81 (D). The images are from single confocal sections ∼1 μm from the bottom of the cell, and selected areas are shown enlarged in the insets. Bars, 10 μm.
Figure Legend Snippet: Uptake of antibodies directed against tetraspanins. MDMs cultured for 13 d were incubated for 3 h at 37°C in medium containing mAbs against CD81, CD53, or CD9 (fed). Cells were then fixed, permeabilized, and stained with antibodies against a different tetraspanin (steady-state). Antibodies were detected with isotype-specific secondary antibodies (red, anti–mouse IgG1-Alexa Fluor 594, for CD81 and CD53; green, anti–mouse IgG2b-Alexa Fluor 488, for CD9). CD9 colocalized with internalized anti-CD81 (A) or anti-CD53 (B). Similarly, internalized anti-CD9 was found in intracellular structures positive for CD53 (C) or CD81 (D). The images are from single confocal sections ∼1 μm from the bottom of the cell, and selected areas are shown enlarged in the insets. Bars, 10 μm.

Techniques Used: Cell Culture, Incubation, Staining

EM analysis of the CD81- and CD9-containing compartment in uninfected MDMs. Ultrathin cryosections from uninfected MDMs (14 d after isolation in A, B, E, and G–I; 26 d after isolation in C, D, F, and J) were stained with mouse mAbs against CD9 (A–G), CD81 (H and I) or CD63 (J), followed by a rabbit anti–mouse IgG bridging antibody and PAG (A–I, 15 nm; J, 10 nm). The black arrow in A marks some small (50 nm diam) vesicles reminiscent of the intralumenal vesicles of MVBs. Black arrowheads in B identify apposed membrane sheets, and the white arrowheads in D show a thick coating on some of these apposed membrane sheets. The white arrow marks a coated pit. Su, cell surface; N, nucleus. Bars: (A, C, and E–J) 200 nm; (B and D) 500 nm.
Figure Legend Snippet: EM analysis of the CD81- and CD9-containing compartment in uninfected MDMs. Ultrathin cryosections from uninfected MDMs (14 d after isolation in A, B, E, and G–I; 26 d after isolation in C, D, F, and J) were stained with mouse mAbs against CD9 (A–G), CD81 (H and I) or CD63 (J), followed by a rabbit anti–mouse IgG bridging antibody and PAG (A–I, 15 nm; J, 10 nm). The black arrow in A marks some small (50 nm diam) vesicles reminiscent of the intralumenal vesicles of MVBs. Black arrowheads in B identify apposed membrane sheets, and the white arrowheads in D show a thick coating on some of these apposed membrane sheets. The white arrow marks a coated pit. Su, cell surface; N, nucleus. Bars: (A, C, and E–J) 200 nm; (B and D) 500 nm.

Techniques Used: Isolation, Staining

Colocalization of HIV with CD63, CD81, or CD9. Primary human MDMs infected with HIV for 10 d were fixed and stained with mouse mAbs against CD63 (A–E), CD81 (F–J), or CD9 (K–O) and rabbit anti-HIV p17, followed by secondary anti–mouse Alexa Fluor 594 and anti–rabbit Alexa Fluor 488 antibodies, and analyzed by confocal microscopy. A–C, F–H, and K–M show projections of 11, 20, and 21 confocal sections, respectively, whereas the images in the two right-hand columns are 0.5-μm-thick confocal sections from the center (D, I, and N) or near the bottom of the cells (E, J, and O). CD63 was found in an extensive network of tubules and vesicles, and also stained the focus of viral p17 (A–D). CD81 and CD9 strongly decorated the cell surface (F and K), but also intracellular puncta near the bottom of the cells (J and O). On medial sections (I and N), the CD81 and CD9 labeling overlapped with the HIV-containing compartment. The asterisk in H marks HIV staining in a syncytium nearby. Bars, 10 μm.
Figure Legend Snippet: Colocalization of HIV with CD63, CD81, or CD9. Primary human MDMs infected with HIV for 10 d were fixed and stained with mouse mAbs against CD63 (A–E), CD81 (F–J), or CD9 (K–O) and rabbit anti-HIV p17, followed by secondary anti–mouse Alexa Fluor 594 and anti–rabbit Alexa Fluor 488 antibodies, and analyzed by confocal microscopy. A–C, F–H, and K–M show projections of 11, 20, and 21 confocal sections, respectively, whereas the images in the two right-hand columns are 0.5-μm-thick confocal sections from the center (D, I, and N) or near the bottom of the cells (E, J, and O). CD63 was found in an extensive network of tubules and vesicles, and also stained the focus of viral p17 (A–D). CD81 and CD9 strongly decorated the cell surface (F and K), but also intracellular puncta near the bottom of the cells (J and O). On medial sections (I and N), the CD81 and CD9 labeling overlapped with the HIV-containing compartment. The asterisk in H marks HIV staining in a syncytium nearby. Bars, 10 μm.

Techniques Used: Infection, Staining, Confocal Microscopy, Labeling

Immunolocalization of the HIV envelope glycoprotein in MDMs. (A) MDMs infected with HIV for 10 d were fixed, permeabilized, and stained with mouse mAbs against the HIV capsid protein p24 and F(ab′) 2 fragments of the human anti-Env mAb 2G12, followed by anti–mouse Alexa Fluor 594 (red) and anti–human FITC (green), respectively. (B) Alternatively, the cells were stained with anti-CD81 and -Env F(ab′) 2 . Env colocalized with assembling virus particles (p24), as well as with CD81. (C and D) Uninfected MDMs were nucleofected with pSVIII HxB2 Env and the distribution of Env was analyzed 48 h after nucleofection, and after treatment with sodium butyrate to boost expression. Fixed and permeabilized cells were double labeled with anti-Env F(ab′) 2 fragments and antibodies against CD81 (C) or CD63 (D) and secondary antibodies as in A. The images show single confocal sections (0.5 μm thick). Bars, 10 μm.
Figure Legend Snippet: Immunolocalization of the HIV envelope glycoprotein in MDMs. (A) MDMs infected with HIV for 10 d were fixed, permeabilized, and stained with mouse mAbs against the HIV capsid protein p24 and F(ab′) 2 fragments of the human anti-Env mAb 2G12, followed by anti–mouse Alexa Fluor 594 (red) and anti–human FITC (green), respectively. (B) Alternatively, the cells were stained with anti-CD81 and -Env F(ab′) 2 . Env colocalized with assembling virus particles (p24), as well as with CD81. (C and D) Uninfected MDMs were nucleofected with pSVIII HxB2 Env and the distribution of Env was analyzed 48 h after nucleofection, and after treatment with sodium butyrate to boost expression. Fixed and permeabilized cells were double labeled with anti-Env F(ab′) 2 fragments and antibodies against CD81 (C) or CD63 (D) and secondary antibodies as in A. The images show single confocal sections (0.5 μm thick). Bars, 10 μm.

Techniques Used: Infection, Staining, Expressing, Labeling

11) Product Images from "Increased Brain-Specific MiR-9 and MiR-124 in the Serum Exosomes of Acute Ischemic Stroke Patients"

Article Title: Increased Brain-Specific MiR-9 and MiR-124 in the Serum Exosomes of Acute Ischemic Stroke Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163645

Characterization of isolated exosomes from human serum. (A) The TEM image shows the spherical morphology of exosomes with a diameter of approximately 100 nm, bar = 200 nm. Representative images show the size distribution (B) and morphology (C) of serum exosomes measured by NTA. (D) Western blotting analysis of exosome markers, CD9, CD63 and CD81, in the serum exosomes from non-stroke controls and stroke patients.
Figure Legend Snippet: Characterization of isolated exosomes from human serum. (A) The TEM image shows the spherical morphology of exosomes with a diameter of approximately 100 nm, bar = 200 nm. Representative images show the size distribution (B) and morphology (C) of serum exosomes measured by NTA. (D) Western blotting analysis of exosome markers, CD9, CD63 and CD81, in the serum exosomes from non-stroke controls and stroke patients.

Techniques Used: Isolation, Transmission Electron Microscopy, Western Blot

12) Product Images from "Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals"

Article Title: Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals

Journal: Journal of Extracellular Vesicles

doi: 10.3402/jev.v4.27378

Isolation of plasma-derived exosomes by size-exclusion chromatography. An aliquot (1 mL) of undiluted plasma from Donor 1 was passed through a sepharose (CL-2B) column, and 30 fractions of 0.5 mL each were collected. (a) SDS–PAGE stained with silver and protein concentration values of fractions 6–14 were measured by Bradford assay (fractions 6, 7 and 8 were below the lower limit of detection) and fractions 6–9 were analysed by flow cytometry, after coupling of vesicles to 4 µm latex beads, for the presence of antigens CD9 (1:10) and CD81 (1:10). The secondary anti-mouse antibody was conjugated to FITC and was used at a 1:100 dilution. MFI: mean fluorescence intensity. (b) Fractions 7–11 were submitted to NTA on a NanoSight LM10 (software version 3.0).
Figure Legend Snippet: Isolation of plasma-derived exosomes by size-exclusion chromatography. An aliquot (1 mL) of undiluted plasma from Donor 1 was passed through a sepharose (CL-2B) column, and 30 fractions of 0.5 mL each were collected. (a) SDS–PAGE stained with silver and protein concentration values of fractions 6–14 were measured by Bradford assay (fractions 6, 7 and 8 were below the lower limit of detection) and fractions 6–9 were analysed by flow cytometry, after coupling of vesicles to 4 µm latex beads, for the presence of antigens CD9 (1:10) and CD81 (1:10). The secondary anti-mouse antibody was conjugated to FITC and was used at a 1:100 dilution. MFI: mean fluorescence intensity. (b) Fractions 7–11 were submitted to NTA on a NanoSight LM10 (software version 3.0).

Techniques Used: Isolation, Derivative Assay, Size-exclusion Chromatography, SDS Page, Staining, Protein Concentration, Bradford Assay, Flow Cytometry, Cytometry, Fluorescence, Software

CD5L and LGALS3BP have similar elution profiles to the exosome marker CD81. Plasma from “Donor 1” was submitted to size-exclusion chromatography and fractions 6–12 were analysed by NTA, flow cytometry and transmission electron microscopy (TEM). (a) NTA was performed on a NanoSight LM10 (software version 3.0). For flow cytometry, samples were coupled to 4 µm beads and incubated with primary antibodies against CD81 (1:10), CD5L (1:100) or LGALS3BP (1:1,000). The secondary antibody was conjugated to Alexa 488 was used at a 1:1,000 dilution. MFI: mean fluorescence intensity. (b) Fraction 6 from size-exclusion chromatography was submitted to cryo-EM and (c) immunostained with anti-CD5L antibodies conjugated to gold spheres of 20 nm.
Figure Legend Snippet: CD5L and LGALS3BP have similar elution profiles to the exosome marker CD81. Plasma from “Donor 1” was submitted to size-exclusion chromatography and fractions 6–12 were analysed by NTA, flow cytometry and transmission electron microscopy (TEM). (a) NTA was performed on a NanoSight LM10 (software version 3.0). For flow cytometry, samples were coupled to 4 µm beads and incubated with primary antibodies against CD81 (1:10), CD5L (1:100) or LGALS3BP (1:1,000). The secondary antibody was conjugated to Alexa 488 was used at a 1:1,000 dilution. MFI: mean fluorescence intensity. (b) Fraction 6 from size-exclusion chromatography was submitted to cryo-EM and (c) immunostained with anti-CD5L antibodies conjugated to gold spheres of 20 nm.

Techniques Used: Marker, Size-exclusion Chromatography, Flow Cytometry, Cytometry, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Software, Incubation, Fluorescence

13) Product Images from "Exosome-mediated transfer of lncRNA-SNHG14 promotes trastuzumab chemoresistance in breast cancer"

Article Title: Exosome-mediated transfer of lncRNA-SNHG14 promotes trastuzumab chemoresistance in breast cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4467

Characterization of exosomes released from trastuzumab-resistant and -sensitive SKBR-3 cells. (A) Transmission electron microscopy images of the exosomes released by SKBR-3/Pr and SKBR-3/Tr cells. (B) Nanoparticle tracking analysis on an LM10 Nanosight unit demonstrating a mean size of 100 nm for SKBR-3/Tr and 120 nm for SKBR-3/Pr exosomes. The size distribution and relative concentration were calculated using the Nano-sight software. (C) Exosomal protein marker (CD63 and CD81) detection by western blotting from purified exosomes and cell extracts. (D) Flow cytometric analysis of the MFI for a panel of exosomal markers: CD9, CD63, CD81 and Alix. Data are presented as the median ± interquartile range of triplicate experiments. MFI, mean fluorescence intensity; CD, cluster of differentiation; Alix, programmed cell death 6-interacting protein.
Figure Legend Snippet: Characterization of exosomes released from trastuzumab-resistant and -sensitive SKBR-3 cells. (A) Transmission electron microscopy images of the exosomes released by SKBR-3/Pr and SKBR-3/Tr cells. (B) Nanoparticle tracking analysis on an LM10 Nanosight unit demonstrating a mean size of 100 nm for SKBR-3/Tr and 120 nm for SKBR-3/Pr exosomes. The size distribution and relative concentration were calculated using the Nano-sight software. (C) Exosomal protein marker (CD63 and CD81) detection by western blotting from purified exosomes and cell extracts. (D) Flow cytometric analysis of the MFI for a panel of exosomal markers: CD9, CD63, CD81 and Alix. Data are presented as the median ± interquartile range of triplicate experiments. MFI, mean fluorescence intensity; CD, cluster of differentiation; Alix, programmed cell death 6-interacting protein.

Techniques Used: Transmission Assay, Electron Microscopy, Concentration Assay, Software, Marker, Western Blot, Purification, Flow Cytometry, Fluorescence

14) Product Images from "Circulating exosomal microRNAs reveal the mechanism of Fructus Meliae Toosendan-induced liver injury in mice"

Article Title: Circulating exosomal microRNAs reveal the mechanism of Fructus Meliae Toosendan-induced liver injury in mice

Journal: Scientific Reports

doi: 10.1038/s41598-018-21113-6

( a ) The size distribution of the serum exosomes was determined using dynamic light scattering (left). Transmission electron micrograph of serum exosomes (middle). The scale bar is 200 nm. The expressions of exosomal marker proteins TSG101 and CD81 were determined using western blot (right). Full-length western blot images in ( a ) are presented in Supplementary Fig. S1 . ( b ) The differentially expressed miRNAs in serum exosomes with the administration of FMT. ( c ) HCA showed two main branches with the DEMs in serum exosomes. ( d ) Real-time quantitative PCR was applied to validate the results of microarray analysis. Gray bars represent microarray data. Black bars indicate the results of real-time quantitative PCR from three technical replicates. Data are presented as the mean fold change ± standard deviation (SD).
Figure Legend Snippet: ( a ) The size distribution of the serum exosomes was determined using dynamic light scattering (left). Transmission electron micrograph of serum exosomes (middle). The scale bar is 200 nm. The expressions of exosomal marker proteins TSG101 and CD81 were determined using western blot (right). Full-length western blot images in ( a ) are presented in Supplementary Fig. S1 . ( b ) The differentially expressed miRNAs in serum exosomes with the administration of FMT. ( c ) HCA showed two main branches with the DEMs in serum exosomes. ( d ) Real-time quantitative PCR was applied to validate the results of microarray analysis. Gray bars represent microarray data. Black bars indicate the results of real-time quantitative PCR from three technical replicates. Data are presented as the mean fold change ± standard deviation (SD).

Techniques Used: Transmission Assay, Marker, Western Blot, High Content Screening, Real-time Polymerase Chain Reaction, Microarray, Standard Deviation

15) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.104836

Fluorescent intensity ratio of CLDN1 and CD81 in 293T cells. 293T cells were transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged g.CD81-r.CD81 ( A ); g.CLDN1-r.CD81 ( B ), or g.CLDN1-r.CLDN1 ( C ). AcGFP and DsRed arbitrary fluorescence units (AFUs) at the cell periphery were determined by laser scanning confocal microscopy and used to generate a scatter plot, allowing one to calculate a correlation coefficient ( r 2 ) and fluorescent intensity ratio (FIR) for each cell analyzed. A representative scatter plot is depicted in the middle column , and the r 2 correlation coefficient ( white bars ) and FIR ( black bars ) values for ten cells are summarized as a bar chart in the final column , where each bar depicts a single cell, and the arrow denotes the respective values for the presented image and scatter plots. In summary, the median FIR values from analyzing ten cells expressing g.CD81-r.CD81, g.CLDN1-r.CD81, and g.CLDN1-r.CLDN1 were 0.56 (IQR 0.53–0.59 and r 2 0.47 (IQR 0.38–0.51)), 0.68 (IQR 0.63–0.74 and r 2 0.45 (IQR 0.39–0.57)), and 0.56 (IQR 0.47–0.64 and r 2 0.29 (IQR 0.25–0.42)), respectively.
Figure Legend Snippet: Fluorescent intensity ratio of CLDN1 and CD81 in 293T cells. 293T cells were transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged g.CD81-r.CD81 ( A ); g.CLDN1-r.CD81 ( B ), or g.CLDN1-r.CLDN1 ( C ). AcGFP and DsRed arbitrary fluorescence units (AFUs) at the cell periphery were determined by laser scanning confocal microscopy and used to generate a scatter plot, allowing one to calculate a correlation coefficient ( r 2 ) and fluorescent intensity ratio (FIR) for each cell analyzed. A representative scatter plot is depicted in the middle column , and the r 2 correlation coefficient ( white bars ) and FIR ( black bars ) values for ten cells are summarized as a bar chart in the final column , where each bar depicts a single cell, and the arrow denotes the respective values for the presented image and scatter plots. In summary, the median FIR values from analyzing ten cells expressing g.CD81-r.CD81, g.CLDN1-r.CD81, and g.CLDN1-r.CLDN1 were 0.56 (IQR 0.53–0.59 and r 2 0.47 (IQR 0.38–0.51)), 0.68 (IQR 0.63–0.74 and r 2 0.45 (IQR 0.39–0.57)), and 0.56 (IQR 0.47–0.64 and r 2 0.29 (IQR 0.25–0.42)), respectively.

Techniques Used: Transfection, Fluorescence, Confocal Microscopy, Expressing

Anti-CD81 modulation of CD81-CD81 and CLDN1-CD81 association(s). 293T cells were transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged g.CD81-r.CD81 or g.CLDN1-r.CD81 and were treated with control anti-VAP1, anti-CD81 mAbs 2s20 and 2s66, 2s66 FAb fragment, and anti-CD9 TS9 at equimolar concentrations (13 μ m ) for 1 h at 37 °C. Representative g.CD81-r.CD81 and g.CLDN1-r.CD81 scatter plots of transfected cells treated with anti-VAP-1, anti-CD81 2s66 IgG, and FAb are shown ( A ). The effect of mAb treatments on g.CD81-r.CD81 ( B ) and g.CLDN1-r.CD81 ( C ) mean correlation coefficient ( r 2 ) and fluorescent intensity ratio (FIR) of ten cells is presented. One way analysis of variance and Dunnett's multiple comparison test were used to determine the degree of significance (*, p
Figure Legend Snippet: Anti-CD81 modulation of CD81-CD81 and CLDN1-CD81 association(s). 293T cells were transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged g.CD81-r.CD81 or g.CLDN1-r.CD81 and were treated with control anti-VAP1, anti-CD81 mAbs 2s20 and 2s66, 2s66 FAb fragment, and anti-CD9 TS9 at equimolar concentrations (13 μ m ) for 1 h at 37 °C. Representative g.CD81-r.CD81 and g.CLDN1-r.CD81 scatter plots of transfected cells treated with anti-VAP-1, anti-CD81 2s66 IgG, and FAb are shown ( A ). The effect of mAb treatments on g.CD81-r.CD81 ( B ) and g.CLDN1-r.CD81 ( C ) mean correlation coefficient ( r 2 ) and fluorescent intensity ratio (FIR) of ten cells is presented. One way analysis of variance and Dunnett's multiple comparison test were used to determine the degree of significance (*, p

Techniques Used: Transfection, Fluorescence

Effect of mutations in CLDN1 and CLDN7 EC1 on CD81 and Occludin association. 293T cells were transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged wild-type and mutant forms of g.CLDN and r.CD81 ( A ) or r.Occludin ( B ), and the degree of association between fluorophore-tagged proteins was assessed by FIR and FRET analysis. C , 293T cells were transfected with AcGFP- and DsRED-tagged versions of wild-type and mutant CLDN constructs to assess the effect of EC1 mutations on CLDN-CLDN cis-interactions. Median FIR and FRET values from ten individual cells are presented (*, p
Figure Legend Snippet: Effect of mutations in CLDN1 and CLDN7 EC1 on CD81 and Occludin association. 293T cells were transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged wild-type and mutant forms of g.CLDN and r.CD81 ( A ) or r.Occludin ( B ), and the degree of association between fluorophore-tagged proteins was assessed by FIR and FRET analysis. C , 293T cells were transfected with AcGFP- and DsRED-tagged versions of wild-type and mutant CLDN constructs to assess the effect of EC1 mutations on CLDN-CLDN cis-interactions. Median FIR and FRET values from ten individual cells are presented (*, p

Techniques Used: Transfection, Fluorescence, Mutagenesis, Construct

Analysis of CLDN-CD81 interactions. 293T cells were transfected to express DsRED-CD81 (r.CD81) and a panel of AcGFP-tagged CLDN (g.CLDN) constructs (CLDN1–17, panels A–I ). Ten cells were imaged as described in Fig. 1 , and estimates of r.CD81 association with g.CLDNs were evaluated by regression analysis and summarized in Table 1 . The images represent scatter plots closest to the median FIR for each g.CLDN-r.CD81 studied.
Figure Legend Snippet: Analysis of CLDN-CD81 interactions. 293T cells were transfected to express DsRED-CD81 (r.CD81) and a panel of AcGFP-tagged CLDN (g.CLDN) constructs (CLDN1–17, panels A–I ). Ten cells were imaged as described in Fig. 1 , and estimates of r.CD81 association with g.CLDNs were evaluated by regression analysis and summarized in Table 1 . The images represent scatter plots closest to the median FIR for each g.CLDN-r.CD81 studied.

Techniques Used: Transfection, Construct

Effect of cell polarization on CLDN1-CD81 and CLDN1-CLDN1 association. HepG2 cells transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged g.CLDN-r.CD81 and g.CLDN1-r.CLDN1 were allowed to polarize over a period of 3 days. Apical bile canalicular structures were identified by staining with anti-ZO-1 and visualized with alexa-633-conjugated secondary anti-rabbit Ig ( A ). Representative scatter plots of g.CLDN-r.CD81 and g.CLDN1-r.CLDN1 at basolateral ( B ) and tight junction ( C ) membrane domains are shown, and the cumulative data from ten cells are summarized below ( D ).
Figure Legend Snippet: Effect of cell polarization on CLDN1-CD81 and CLDN1-CLDN1 association. HepG2 cells transfected to express AcGFP ( g ) and DsRED ( r ) fluorescence-tagged g.CLDN-r.CD81 and g.CLDN1-r.CLDN1 were allowed to polarize over a period of 3 days. Apical bile canalicular structures were identified by staining with anti-ZO-1 and visualized with alexa-633-conjugated secondary anti-rabbit Ig ( A ). Representative scatter plots of g.CLDN-r.CD81 and g.CLDN1-r.CLDN1 at basolateral ( B ) and tight junction ( C ) membrane domains are shown, and the cumulative data from ten cells are summarized below ( D ).

Techniques Used: Transfection, Fluorescence, Staining

Analysis of CLDN1-CD81 extracellular loop interactions by surface plasmon resonance. MBP-CLDN1 EC1 ( A ) and MBP-CD81 EC2 ( B and C ) were immobilized onto the bio-sensor chip surface. Homotypic protein interactions were demonstrated by flowing MBP-CLDN1 EC1 ( solid gray line ) and MBP-CD81 EC2 ( solid black line ) over the respective chip surfaces ( A and B ) with both MBP-CLDN7 EC1 ( dotted light gray line ) and MBP ( dotted black line ) as negative controls at a concentration of 1 mg/ml. Heterotypic interaction between MBP-CLDN1-EC1 and MBP-CD81 EC2 is depicted in C . To control for nonspecific interactions, all MBP fusion proteins were flowed over an activated and blocked “empty” channel, and the response unit(s) were subtracted from the test channels. The arrow indicates the “association time” i when proteins are flowed over the respective chip surfaces and the “dissociation phase” begins at time ii when protein injection is stopped. Data are representative of two independent experiments.
Figure Legend Snippet: Analysis of CLDN1-CD81 extracellular loop interactions by surface plasmon resonance. MBP-CLDN1 EC1 ( A ) and MBP-CD81 EC2 ( B and C ) were immobilized onto the bio-sensor chip surface. Homotypic protein interactions were demonstrated by flowing MBP-CLDN1 EC1 ( solid gray line ) and MBP-CD81 EC2 ( solid black line ) over the respective chip surfaces ( A and B ) with both MBP-CLDN7 EC1 ( dotted light gray line ) and MBP ( dotted black line ) as negative controls at a concentration of 1 mg/ml. Heterotypic interaction between MBP-CLDN1-EC1 and MBP-CD81 EC2 is depicted in C . To control for nonspecific interactions, all MBP fusion proteins were flowed over an activated and blocked “empty” channel, and the response unit(s) were subtracted from the test channels. The arrow indicates the “association time” i when proteins are flowed over the respective chip surfaces and the “dissociation phase” begins at time ii when protein injection is stopped. Data are representative of two independent experiments.

Techniques Used: SPR Assay, Chromatin Immunoprecipitation, Concentration Assay, Injection

16) Product Images from "Circulating Exosomal microRNAs as Biomarkers of Systemic Lupus Erythematosus"

Article Title: Circulating Exosomal microRNAs as Biomarkers of Systemic Lupus Erythematosus

Journal: Clinics

doi: 10.6061/clinics/2020/e1528

Western blotting analysis of the exosomal preparations showing the expression of the exosomal markers CD63, CD81, and TSG101.
Figure Legend Snippet: Western blotting analysis of the exosomal preparations showing the expression of the exosomal markers CD63, CD81, and TSG101.

Techniques Used: Western Blot, Expressing

17) Product Images from "Exosomal miR-19a from adipose-derived stem cells suppresses differentiation of corneal keratocytes into myofibroblasts"

Article Title: Exosomal miR-19a from adipose-derived stem cells suppresses differentiation of corneal keratocytes into myofibroblasts

Journal: Aging (Albany NY)

doi: 10.18632/aging.102802

Characterization of ADSCs and ADSCs-Exo. ( A , B ) Flow cytometry analysis of ADSCs isolated from rabbit adipose tissues is shown using fluorescent-tagged antibodies against cell surface proteins, namely, CD29, CD90, CD34, and CD45. ( C ) The mean diameter of ADSCs exosomes was analyzed using a nanoparticle tracking system (NTA). NTA analysis of the exosomes isolated from ADSCs (ADSCs-Exo) shows a mean concentration of 1.1 x 10 10 particles per mL. ( D , E ) Western blot analysis shows levels of CD9, CD81 and flotillin-1 proteins in the ADSCs and the ADSCs-Exo. GAPDH was used as an internal control. The levels of CD9, CD81 and flotillin-1 are expressed relative to GAPDH. ** denotes P
Figure Legend Snippet: Characterization of ADSCs and ADSCs-Exo. ( A , B ) Flow cytometry analysis of ADSCs isolated from rabbit adipose tissues is shown using fluorescent-tagged antibodies against cell surface proteins, namely, CD29, CD90, CD34, and CD45. ( C ) The mean diameter of ADSCs exosomes was analyzed using a nanoparticle tracking system (NTA). NTA analysis of the exosomes isolated from ADSCs (ADSCs-Exo) shows a mean concentration of 1.1 x 10 10 particles per mL. ( D , E ) Western blot analysis shows levels of CD9, CD81 and flotillin-1 proteins in the ADSCs and the ADSCs-Exo. GAPDH was used as an internal control. The levels of CD9, CD81 and flotillin-1 are expressed relative to GAPDH. ** denotes P

Techniques Used: Flow Cytometry, Isolation, Concentration Assay, Western Blot

18) Product Images from "Circulating Extracellular Vesicle Proteins and MicroRNA Profiles in Subcortical and Cortical-Subcortical Ischaemic Stroke"

Article Title: Circulating Extracellular Vesicle Proteins and MicroRNA Profiles in Subcortical and Cortical-Subcortical Ischaemic Stroke

Journal: Biomedicines

doi: 10.3390/biomedicines9070786

Participants, EV characterisation, and EV levels: ( A ) number of patients enrolled in the study; ( B ) characteristics of EVs. Size and concentration of the particles detected in the sample EVs isolated from HCs by NanoSight; ( C ) electron microscope image. EVs from HCs smaller than 100 nm were observed by electron microscope; ( D ) Western blot. Detection of EVs from HCs with specific markers (positive: Alix, CD63 and CD81; negative: Albumin) by Western blot. Negative control samples are serum. The gel image was cropped; ( E ) EV levels at 24 h showed no differences between the CSC, SC, and HC groups; ( F ) correlation between EV levels and total infarct volume. Data are expressed as mean ± SD. Abbreviations: CSC, cortical-subcortical stroke; EVs, extracellular vesicles; HCs, healthy controls; SC, subcortical stroke. The samples were run after only one freezing.
Figure Legend Snippet: Participants, EV characterisation, and EV levels: ( A ) number of patients enrolled in the study; ( B ) characteristics of EVs. Size and concentration of the particles detected in the sample EVs isolated from HCs by NanoSight; ( C ) electron microscope image. EVs from HCs smaller than 100 nm were observed by electron microscope; ( D ) Western blot. Detection of EVs from HCs with specific markers (positive: Alix, CD63 and CD81; negative: Albumin) by Western blot. Negative control samples are serum. The gel image was cropped; ( E ) EV levels at 24 h showed no differences between the CSC, SC, and HC groups; ( F ) correlation between EV levels and total infarct volume. Data are expressed as mean ± SD. Abbreviations: CSC, cortical-subcortical stroke; EVs, extracellular vesicles; HCs, healthy controls; SC, subcortical stroke. The samples were run after only one freezing.

Techniques Used: Concentration Assay, Isolation, Microscopy, Western Blot, Negative Control

19) Product Images from "Exosomes derived from human umbilical cord mesenchymal stem cells alleviate inflammatory bowel disease in mice through ubiquitination"

Article Title: Exosomes derived from human umbilical cord mesenchymal stem cells alleviate inflammatory bowel disease in mice through ubiquitination

Journal: American Journal of Translational Research

doi:

Characterization of exosomes derived from human umbilical cord mesenchymal stem cells. A. Transmission electron microscopy to identify the characteristics of hUCMSC-exosomes. Scale bar: 100 nm. B. Nanoparticle Tracking Analysis (NAT) of exosomes. C. CD9, CD63, and CD81 expressions of exosomes are detected using western blot assay.
Figure Legend Snippet: Characterization of exosomes derived from human umbilical cord mesenchymal stem cells. A. Transmission electron microscopy to identify the characteristics of hUCMSC-exosomes. Scale bar: 100 nm. B. Nanoparticle Tracking Analysis (NAT) of exosomes. C. CD9, CD63, and CD81 expressions of exosomes are detected using western blot assay.

Techniques Used: Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot

20) Product Images from "Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice"

Article Title: Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice

Journal: Stem Cell Research & Therapy

doi: 10.1186/scrt546

Efficient differentiation of mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSCs) and characterization of iMSCs-Exo. (A) Phase-contrast image of long-term cultured iPSC clone before differentiation (i), intermediate phase of induced iPSCs (ii), and iMSCs (iii). (B) Quantitative reverse-transcriptase polymerase chain reaction analysis of the expression level of pluripotent genes (Nanog, Oct4, and Msx1) in iPSCs during differentiation (i) and in iPSCs and iMSCs (ii). (C) Flow cytometric analysis of mesenchymal positive markers, such as CD29, CD44, CD73, CD90, CD105, and CD146, and negative markers, such as CD34, CD45, CD133, and HLA-DR. Black histograms represent the isotype controls, and the red solid peak represents the marker indicated. (D) Multi-differentiation potential of iMSCs. (i) Alizarin Red staining of osteogenic mineralization (day 14). (ii) Oil Red O staining of small lipid droplets (day 21). (iii) Toluidine Blue staining of cartilaginous extracellular matrix (day 21). (E) Morphology of iMSCs-Exo under transmission electron microscopy. (F) Western blotting analysis of exosomal CD63, CD81, and CD9 protein in iMSCs and iMSCs-Exo. The culture medium served as the control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iMSC, induced pluripotent stem cell-derived mesenchymal stem cell; iMSCs-Exo, exosomes derived from induced pluripotent stem cell-derived mesenchymal stem cells.
Figure Legend Snippet: Efficient differentiation of mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSCs) and characterization of iMSCs-Exo. (A) Phase-contrast image of long-term cultured iPSC clone before differentiation (i), intermediate phase of induced iPSCs (ii), and iMSCs (iii). (B) Quantitative reverse-transcriptase polymerase chain reaction analysis of the expression level of pluripotent genes (Nanog, Oct4, and Msx1) in iPSCs during differentiation (i) and in iPSCs and iMSCs (ii). (C) Flow cytometric analysis of mesenchymal positive markers, such as CD29, CD44, CD73, CD90, CD105, and CD146, and negative markers, such as CD34, CD45, CD133, and HLA-DR. Black histograms represent the isotype controls, and the red solid peak represents the marker indicated. (D) Multi-differentiation potential of iMSCs. (i) Alizarin Red staining of osteogenic mineralization (day 14). (ii) Oil Red O staining of small lipid droplets (day 21). (iii) Toluidine Blue staining of cartilaginous extracellular matrix (day 21). (E) Morphology of iMSCs-Exo under transmission electron microscopy. (F) Western blotting analysis of exosomal CD63, CD81, and CD9 protein in iMSCs and iMSCs-Exo. The culture medium served as the control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iMSC, induced pluripotent stem cell-derived mesenchymal stem cell; iMSCs-Exo, exosomes derived from induced pluripotent stem cell-derived mesenchymal stem cells.

Techniques Used: Cell Culture, Polymerase Chain Reaction, Expressing, Flow Cytometry, Marker, Staining, Transmission Assay, Electron Microscopy, Western Blot, Derivative Assay

21) Product Images from "Extracellular vesicles derived from human dental pulp stem cells promote osteogenesis of adipose-derived stem cells via the MAPK pathway"

Article Title: Extracellular vesicles derived from human dental pulp stem cells promote osteogenesis of adipose-derived stem cells via the MAPK pathway

Journal: Journal of Tissue Engineering

doi: 10.1177/2041731420975569

Characteristics of hDPSC-EVs. (a) TEM observation of hDPSC-EVs; (b) particle size identification of hDPSC-EVs detected by NTA; (c) western blotting analysis of CD9, CD63, CD81, and TSG101 in EVs.
Figure Legend Snippet: Characteristics of hDPSC-EVs. (a) TEM observation of hDPSC-EVs; (b) particle size identification of hDPSC-EVs detected by NTA; (c) western blotting analysis of CD9, CD63, CD81, and TSG101 in EVs.

Techniques Used: Transmission Electron Microscopy, Western Blot

22) Product Images from "Multiple-Cycle Polymeric Extracellular Vesicle Precipitation and Its Evaluation by Targeted Mass Spectrometry"

Article Title: Multiple-Cycle Polymeric Extracellular Vesicle Precipitation and Its Evaluation by Targeted Mass Spectrometry

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22094311

The purity evaluation. ( A ) Western blot analysis of CD9, CD63, and CD81 on 1–4-cycle EV fractions. ( B ) The ALB levels measured in EV fractions (1–4 cycles) and UC using an MRM assay. ( C ) The levels of 3 EV markers (A2M, LGALS3BP, and THBS 1) and ALB in non-purified and EV-purified fractions (2-cycle) obtained by an MRM assay. * , p -value
Figure Legend Snippet: The purity evaluation. ( A ) Western blot analysis of CD9, CD63, and CD81 on 1–4-cycle EV fractions. ( B ) The ALB levels measured in EV fractions (1–4 cycles) and UC using an MRM assay. ( C ) The levels of 3 EV markers (A2M, LGALS3BP, and THBS 1) and ALB in non-purified and EV-purified fractions (2-cycle) obtained by an MRM assay. * , p -value

Techniques Used: Western Blot, MRM Assay, Purification

23) Product Images from "An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification"

Article Title: An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification

Journal: Theranostics

doi: 10.7150/thno.49047

Comparison of the apta-ELISA, apta-HCR-ELISA and apta-HCR-CRISPR assays. (A) apta-ELISA mechanism. In the apta-ELISA assay, EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in streptavidin-HRP. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (B) Detection of nucleolin + EVs by apta-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (C) apta-ELISA-HCR mechanism. EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in premixture HRP-labeled H1 and H2. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (D) Detection of nucleolin + EVs by apta-HCR-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (E) apta-HCR-CRISPR mechanism. Based on the apta-ELISA-HCR assay, the HCR products were targeted by Cas12a/crRNA duplex and triggered Cas12a to cleave the ssDNA-FQ reporter substrate, resulting in readable and accumulating FI proportional to the concentration of target positive EVs. (F) Detection of nucleolin + EVs by apta-HCR- CRISPR with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (G) Comparison of the LOD of apta-HCR-CRISPR, apta-HCR-ELISA and apta-ELISA in detecting nucleolin + EV spiked in PBS. (H) The concentration change in nucleolin + EVs is linearly related to the FI through fitting curves, Y= 7663 lg (EVs) - 12852 (R 2 = 0.9848). FI, fluorescence intensity. PBS served as a blank. The P values were calculated using a one-way ANOVA followed by a Sidak multiple-comparison with the former group. *, **, *** and **** represent P
Figure Legend Snippet: Comparison of the apta-ELISA, apta-HCR-ELISA and apta-HCR-CRISPR assays. (A) apta-ELISA mechanism. In the apta-ELISA assay, EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in streptavidin-HRP. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (B) Detection of nucleolin + EVs by apta-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (C) apta-ELISA-HCR mechanism. EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in premixture HRP-labeled H1 and H2. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (D) Detection of nucleolin + EVs by apta-HCR-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (E) apta-HCR-CRISPR mechanism. Based on the apta-ELISA-HCR assay, the HCR products were targeted by Cas12a/crRNA duplex and triggered Cas12a to cleave the ssDNA-FQ reporter substrate, resulting in readable and accumulating FI proportional to the concentration of target positive EVs. (F) Detection of nucleolin + EVs by apta-HCR- CRISPR with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (G) Comparison of the LOD of apta-HCR-CRISPR, apta-HCR-ELISA and apta-ELISA in detecting nucleolin + EV spiked in PBS. (H) The concentration change in nucleolin + EVs is linearly related to the FI through fitting curves, Y= 7663 lg (EVs) - 12852 (R 2 = 0.9848). FI, fluorescence intensity. PBS served as a blank. The P values were calculated using a one-way ANOVA followed by a Sidak multiple-comparison with the former group. *, **, *** and **** represent P

Techniques Used: Enzyme-linked Immunosorbent Assay, CRISPR, Incubation, Concentration Assay, Labeling, Host-Cell Reactivation, Fluorescence

Schematic of apta-HCR-CRISPR. The EVs are captured by a cocktail of anti-CD63-, anti-CD81- and anti-CD9 antibody-coated beads and recognized with H0. The formed antibody-EV-H0 complexes trigger HCR and generate long repetitive target sequences that are specifically recognized by the added crRNA/Cas12a duplex. Target-activated Cas12a trans-cleaves nearby ssDNA-FQ reporter, resulting in readable and accumulating fluorescence signal proportional to the concentration of target positive EVs.
Figure Legend Snippet: Schematic of apta-HCR-CRISPR. The EVs are captured by a cocktail of anti-CD63-, anti-CD81- and anti-CD9 antibody-coated beads and recognized with H0. The formed antibody-EV-H0 complexes trigger HCR and generate long repetitive target sequences that are specifically recognized by the added crRNA/Cas12a duplex. Target-activated Cas12a trans-cleaves nearby ssDNA-FQ reporter, resulting in readable and accumulating fluorescence signal proportional to the concentration of target positive EVs.

Techniques Used: CRISPR, Fluorescence, Concentration Assay

24) Product Images from "Organization and regulation of intracellular plasma membrane-connected HIV-1 assembly compartments in macrophages"

Article Title: Organization and regulation of intracellular plasma membrane-connected HIV-1 assembly compartments in macrophages

Journal: BMC Biology

doi: 10.1186/1741-7007-11-89

Association of actin with IPMCs. (A) HIV-infected MDMs were embedded in Epon, sectioned and analyzed by transmission EM, as previously described [ 14 ]. HIV particles are seen in a complex IPMC. Some of the IPMC membranes are covered with a coat of electron-dense focal adhesion proteins (arrowheads) and a layer of fine filaments (black arrows). L, lipid droplets; M, mitochondrion; the asterisk marks a budding HIV particle. Epon EM was conducted on MDMs from two donors. Scale bar = 500 nm. (B) Uninfected MDMs were stained for CD81 and actin (Alexa Fluor 594-conjugated phalloidin). (C) HIV-1-infected MDMs were stained for the matrix protein p17 and actin. The images show single optical sections acquired with a Leica SPE confocal microscope as above. Scale bars = 10 μm.
Figure Legend Snippet: Association of actin with IPMCs. (A) HIV-infected MDMs were embedded in Epon, sectioned and analyzed by transmission EM, as previously described [ 14 ]. HIV particles are seen in a complex IPMC. Some of the IPMC membranes are covered with a coat of electron-dense focal adhesion proteins (arrowheads) and a layer of fine filaments (black arrows). L, lipid droplets; M, mitochondrion; the asterisk marks a budding HIV particle. Epon EM was conducted on MDMs from two donors. Scale bar = 500 nm. (B) Uninfected MDMs were stained for CD81 and actin (Alexa Fluor 594-conjugated phalloidin). (C) HIV-1-infected MDMs were stained for the matrix protein p17 and actin. The images show single optical sections acquired with a Leica SPE confocal microscope as above. Scale bars = 10 μm.

Techniques Used: Infection, Transmission Assay, Staining, Microscopy

Localization of PI ( 4 , 5)P 2 and phosphatidylinositol 3-phosphate in MDMs. MDMs were nucleofected to express PH-GFP, to localize (A , B) PI(4,5)P 2 or (C) a double-FYVE domain construct fused to GFP (2xFYVE-GFP), a probe for phosphatidylinositol 3-phosphate. After 24 hours the cells were fixed and co-stained for CD81 or CD44 as indicated. (D) Seven-day-old MDMs were infected with HIV-1 BaL and after a further 7 days were nucleofected to express PH-GFP, incubated for 24 hours, then fixed and stained for the viral matrix protein p17. Images show single confocal sections, and were acquired with a Leica SPE confocal microscope as in Figure 1 . All experiments were repeated on cells from at least three different donors. Scale bars = 10 μm. PI(4,5)P2: Phosphatidylinositol 4,5-bisphosphate; PH-GFP: Phospholipase Cδ pleckstrin homology domain linked to GFP.
Figure Legend Snippet: Localization of PI ( 4 , 5)P 2 and phosphatidylinositol 3-phosphate in MDMs. MDMs were nucleofected to express PH-GFP, to localize (A , B) PI(4,5)P 2 or (C) a double-FYVE domain construct fused to GFP (2xFYVE-GFP), a probe for phosphatidylinositol 3-phosphate. After 24 hours the cells were fixed and co-stained for CD81 or CD44 as indicated. (D) Seven-day-old MDMs were infected with HIV-1 BaL and after a further 7 days were nucleofected to express PH-GFP, incubated for 24 hours, then fixed and stained for the viral matrix protein p17. Images show single confocal sections, and were acquired with a Leica SPE confocal microscope as in Figure 1 . All experiments were repeated on cells from at least three different donors. Scale bars = 10 μm. PI(4,5)P2: Phosphatidylinositol 4,5-bisphosphate; PH-GFP: Phospholipase Cδ pleckstrin homology domain linked to GFP.

Techniques Used: Construct, Staining, Infection, Incubation, Microscopy

25) Product Images from "Pro inflammatory stimuli enhance the immunosuppressive functions of adipose mesenchymal stem cells-derived exosomes"

Article Title: Pro inflammatory stimuli enhance the immunosuppressive functions of adipose mesenchymal stem cells-derived exosomes

Journal: Scientific Reports

doi: 10.1038/s41598-018-31707-9

Characterization of AMSCs derived–exosomes. ( A ) Immunoblotting of AMSCs-derived exosomes, Exoquick-derived supernatants (SN) and AMSCs lysate for CD9, CD63, CD81 and TSG101 exosomal protein and Calnexin, GRP94 and RISC contaminants (cropped images – uncropped originals available in Supplementary Figure 2 ). ( B ) The concentration of exosomes was quantified measuring the enzymatic activity of the exosomal AChE enzyme by Exocet kit. Particles size were quantified by qNano system ( D ) and a representative graph of frequency size distribution is shown ( C ). Columns, mean; bars, SD.
Figure Legend Snippet: Characterization of AMSCs derived–exosomes. ( A ) Immunoblotting of AMSCs-derived exosomes, Exoquick-derived supernatants (SN) and AMSCs lysate for CD9, CD63, CD81 and TSG101 exosomal protein and Calnexin, GRP94 and RISC contaminants (cropped images – uncropped originals available in Supplementary Figure 2 ). ( B ) The concentration of exosomes was quantified measuring the enzymatic activity of the exosomal AChE enzyme by Exocet kit. Particles size were quantified by qNano system ( D ) and a representative graph of frequency size distribution is shown ( C ). Columns, mean; bars, SD.

Techniques Used: Derivative Assay, Concentration Assay, Activity Assay

26) Product Images from "Intranasally Administered Extracellular Vesicles from Adipose Stem Cells Have Immunomodulatory Effects in a Mouse Model of Asthma"

Article Title: Intranasally Administered Extracellular Vesicles from Adipose Stem Cells Have Immunomodulatory Effects in a Mouse Model of Asthma

Journal: Stem Cells International

doi: 10.1155/2021/6686625

Characterization of adipose stem cell- (ASC-) derived extracellular vesicles (EVs). (a) Transmission electron microscopy of ASC-derived EVs showed a spherical shape and lipid bilayers (original magnification ×250,000; the scale bar indicates 200 nm). (b) Average size of the EVs calculated by dynamic light scattering was 211.7 ± 26.6 nm, and average concentration was 57,946,000 ± 12,519,069 particles/ml. (c) Nanoparticle tracking analysis revealed corresponding EV size distribution and a mode of 127.1 ± 3.2 nm. (d) Western blotting of EVs showed high expression of CD81, CD40, and calnexin.
Figure Legend Snippet: Characterization of adipose stem cell- (ASC-) derived extracellular vesicles (EVs). (a) Transmission electron microscopy of ASC-derived EVs showed a spherical shape and lipid bilayers (original magnification ×250,000; the scale bar indicates 200 nm). (b) Average size of the EVs calculated by dynamic light scattering was 211.7 ± 26.6 nm, and average concentration was 57,946,000 ± 12,519,069 particles/ml. (c) Nanoparticle tracking analysis revealed corresponding EV size distribution and a mode of 127.1 ± 3.2 nm. (d) Western blotting of EVs showed high expression of CD81, CD40, and calnexin.

Techniques Used: Derivative Assay, Transmission Assay, Electron Microscopy, Concentration Assay, Western Blot, Expressing

27) Product Images from "Exosomes Derived from Human Endothelial Progenitor Cells Accelerate Cutaneous Wound Healing by Promoting Angiogenesis Through Erk1/2 Signaling"

Article Title: Exosomes Derived from Human Endothelial Progenitor Cells Accelerate Cutaneous Wound Healing by Promoting Angiogenesis Through Erk1/2 Signaling

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.15514

Characterization of exosomes released by human UCB-derived EPCs (EPC-Exos). (A) Particle size distribution and concentration of EPC-Exos measured by TRPS. (B) Morphology of EPC-Exos under a transmission electron microscopy. Scale bar: 50nm. (C) Western blotting analysis of exosomal surface marker proteins (including CD9, CD63, and CD81) and endothelial lineage cell marker (CD31) in EPC-Exos.
Figure Legend Snippet: Characterization of exosomes released by human UCB-derived EPCs (EPC-Exos). (A) Particle size distribution and concentration of EPC-Exos measured by TRPS. (B) Morphology of EPC-Exos under a transmission electron microscopy. Scale bar: 50nm. (C) Western blotting analysis of exosomal surface marker proteins (including CD9, CD63, and CD81) and endothelial lineage cell marker (CD31) in EPC-Exos.

Techniques Used: Derivative Assay, Concentration Assay, Transmission Assay, Electron Microscopy, Western Blot, Marker

28) Product Images from "Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells"

Article Title: Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006219

Vector particles are associated with tetraspanin-enriched compartments. Jurkat and SupT1 cells were exposed to vector for 1 or 24 hours. Cells were stained with Hoechst 33342 nuclear stain, washed, fixed, permeabilized, cytospun, and stained with anti-CD81 or anti-CD63 antibody (TAPA1, tetraspanin). (A,B) Vector genome association with select tetraspanins. Columns represent total vector number (gray, numerator in each column) over particles colocalized with tetraspanins (black, denominator in each column). Fluorescent images were deconvolved to confirm intracellular particle location. (C) Representative images illustrating vector genome association with tetraspanins. Particles are GFP-vpr labeled (green), CD81 or CD63 tetraspanin is 2°-labeled with Alexa Fluor 647 (far-red). Yellow indicates colocalization of the two fluorescent signals. Only fully merged and overlapping particles were counted as colocalized.
Figure Legend Snippet: Vector particles are associated with tetraspanin-enriched compartments. Jurkat and SupT1 cells were exposed to vector for 1 or 24 hours. Cells were stained with Hoechst 33342 nuclear stain, washed, fixed, permeabilized, cytospun, and stained with anti-CD81 or anti-CD63 antibody (TAPA1, tetraspanin). (A,B) Vector genome association with select tetraspanins. Columns represent total vector number (gray, numerator in each column) over particles colocalized with tetraspanins (black, denominator in each column). Fluorescent images were deconvolved to confirm intracellular particle location. (C) Representative images illustrating vector genome association with tetraspanins. Particles are GFP-vpr labeled (green), CD81 or CD63 tetraspanin is 2°-labeled with Alexa Fluor 647 (far-red). Yellow indicates colocalization of the two fluorescent signals. Only fully merged and overlapping particles were counted as colocalized.

Techniques Used: Plasmid Preparation, Staining, Labeling

Vector particles associate with MVB markers. (A,B) Vector genomes associate with select MVB markers. Jurkat cells exposed to GFP-vpr vector (green) overnight, followed by pronase wash, and stain with antibodies against CD81, CD63, or N-Rh-PE (red), MHCII (magenta). Particles found associated with CD81 and MHCII, or N-Rh-PE and MHCII are white. (C) Live cell imaging of vector-exposed, pronase-washed 1° SupT1 cells (labeled with anti-CD81, Alexa Fluor 647, magenta) in co-culture with 2° 293T DsRed actin (red) expressing cells. Right hand panels lack the DsRed layer for improved visual clarity of otherwise identical frames. Genomes co-localized with tetraspanin are white (arrows, boxes). Genomes co-localized with DsRed actin are yellow. Deconvolution microscopy was performed on live cells by collecting series of z -stacks (0.5 µm) every minute for 10 minutes, elapsed time is indicated in black.
Figure Legend Snippet: Vector particles associate with MVB markers. (A,B) Vector genomes associate with select MVB markers. Jurkat cells exposed to GFP-vpr vector (green) overnight, followed by pronase wash, and stain with antibodies against CD81, CD63, or N-Rh-PE (red), MHCII (magenta). Particles found associated with CD81 and MHCII, or N-Rh-PE and MHCII are white. (C) Live cell imaging of vector-exposed, pronase-washed 1° SupT1 cells (labeled with anti-CD81, Alexa Fluor 647, magenta) in co-culture with 2° 293T DsRed actin (red) expressing cells. Right hand panels lack the DsRed layer for improved visual clarity of otherwise identical frames. Genomes co-localized with tetraspanin are white (arrows, boxes). Genomes co-localized with DsRed actin are yellow. Deconvolution microscopy was performed on live cells by collecting series of z -stacks (0.5 µm) every minute for 10 minutes, elapsed time is indicated in black.

Techniques Used: Plasmid Preparation, Staining, Live Cell Imaging, Labeling, Co-Culture Assay, Expressing, Microscopy

29) Product Images from "Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells"

Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138849

Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P
Figure Legend Snippet: Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

Techniques Used: Electron Microscopy, Isolation, Western Blot, Cell Culture, Activity Assay

Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.
Figure Legend Snippet: Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

Techniques Used: Derivative Assay, Electron Microscopy, Isolation, Western Blot, Cell Culture, SDS Page, Electrophoresis, Staining, Software

30) Product Images from "The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication"

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

Journal: Nature Communications

doi: 10.1038/s41467-019-08299-7

Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles
Figure Legend Snippet: Model of circadian clock components regulating HCV, DENV and ZIKV replication. The circadian activator BMAL1 regulates HCV entry into hepatocytes through modulating viral receptors CD81 and claudin-1 expression. Activating REV-ERB with synthetic agonists or protein overexpression inhibits HCV, DENV or ZIKV RNA replication via modulating SCD and subsequent release of infectious particles

Techniques Used: Expressing, Over Expression

HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)
Figure Legend Snippet: HCV entry is circadian regulated. a Synchronisation of Huh-7 cells. Huh-7 cells were serum shocked and Bmal1 and Rev-Erbα mRNA measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and expressed relative to circadian time (CT) 0. Data are the average of four independent experiments ( n = 4). b Circadian infection protocol. Synchronised Huh-7 were inoculated with HCVpp or HCVcc particles for 1 h and infectivity determined by luciferase assay or measuring the frequency of viral NS5A expressing cells 24 h later. c HCV entry shows a circadian pattern. Synchronised Huh-7 cells were inoculated with HCVpp at defined CTs and particle entry measured 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 8, Kruskal–Wallis ANOVA with Dunn’s test. d HCV infection shows a circadian pattern. Synchronised Huh-7 were inoculated with HCVcc SA13/JFH-1 or J6/JFH-1 at defined CTs and the frequency of infected cells quantified 24 h later and data expressed relative to CT0; mean ± S.E.M., n = 4; Kruskal–Wallis ANOVA with Dunn’s test. e Circadian pattern of HCV receptors. Synchronised Huh-7 were lysed every 8 h, total RNA extracted and CD81 , claudin-1 and occludin mRNA levels together with the housekeeping GAPDH assessed by qPCR. Individual receptor transcripts were normalised to CT0. Representative of three independent experiments; n = 3, mean ± S.E.M. f BMAL1 regulates HCV entry receptors. Parental (WT) or Bmal1 KO Huh-7 lysates were assessed for BMAL1 and viral receptors CD81, claudin-1 and occludin expression together with housekeeping GAPDH by western blotting. Total RNA from parental or Bmal1 KO Huh-7 were extracted and mRNA of CD81, claudin-1, occludin and GAPDH measured by qRT-PCR. Data are expressed relative to parental cells (mean ± S.E.M., n = 3, Mann–Whitney test). g BMAL1 regulates HCV entry and infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with HCVpp or HCVcc SA13/JFH-1 and infection assessed after 24 h. Data are expressed relative to WT cells (mean ± S.E.M., n = 5, Mann–Whitney test). h BMAL1 regulates DENV and ZIKV infection. WT or Bmal1 KO Huh-7 were inoculated for 1 h with DENV or ZIKV and infection assessed after 24 h. Data are expressed relative to WT (mean ± S.E.M., n = 6 for DENV; n = 9 for ZIKV, Mann–Whitney test)

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

REV-ERB agonists inhibit hepatitis C virus (HCV) entry. a REV-ERB agonists inhibit BMAL1 transcription. Huh-7 cells were treated with REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and Bmal1 mRNA levels quantified by qRT-PCR, respectively (mean ± S.E.M., n = 7, Kruskal–Wallis ANOVA with Dunn’s test). b , c REV-ERB agonists modulate HCV receptor expression. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and the cells lysed, total protein measured and assessed for CD81, claudin-1 and occludin expression together with the housekeeping GAPDH by western blotting or mass spectrometric analysis (mean ± S.E.M., n = 3, Mann–Whitney test). d REV-ERB agonists inhibit HCV entry. Huh-7 cells were treated with increasing dose of REV-ERB agonists SR9009 or GSK2667 for 24 h, inoculated with HCVpp and infection assessed 24 h later (mean ± S.E.M., n = 5). e REV-ERB agonists inhibit HCVpp bearing patient-derived glycoproteins. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h, infected with HCVpp bearing patient-derived envelope glycoproteins and infection assessed 24 h later. In all cases, data are expressed relative to untreated (Ctrl) cells. (Mean ± S.E.M., n = 3, Kruskal–Wallis ANOVA)
Figure Legend Snippet: REV-ERB agonists inhibit hepatitis C virus (HCV) entry. a REV-ERB agonists inhibit BMAL1 transcription. Huh-7 cells were treated with REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and Bmal1 mRNA levels quantified by qRT-PCR, respectively (mean ± S.E.M., n = 7, Kruskal–Wallis ANOVA with Dunn’s test). b , c REV-ERB agonists modulate HCV receptor expression. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h and the cells lysed, total protein measured and assessed for CD81, claudin-1 and occludin expression together with the housekeeping GAPDH by western blotting or mass spectrometric analysis (mean ± S.E.M., n = 3, Mann–Whitney test). d REV-ERB agonists inhibit HCV entry. Huh-7 cells were treated with increasing dose of REV-ERB agonists SR9009 or GSK2667 for 24 h, inoculated with HCVpp and infection assessed 24 h later (mean ± S.E.M., n = 5). e REV-ERB agonists inhibit HCVpp bearing patient-derived glycoproteins. Huh-7 cells were treated with the REV-ERB agonists SR9009 or GSK2667 (20 µM) for 24 h, infected with HCVpp bearing patient-derived envelope glycoproteins and infection assessed 24 h later. In all cases, data are expressed relative to untreated (Ctrl) cells. (Mean ± S.E.M., n = 3, Kruskal–Wallis ANOVA)

Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY, Infection, Derivative Assay

REV-ERBα inhibits hepatitis C virus (HCV) RNA replication. a Silencing Rev-erbα increases HCV replication. Huh-7 cells supporting a HCV JFH-1-LUC replicon were transduced with lentivirus encoding sh Rev-erbα or control and silencing confirmed by measuring Rev-erbα mRNA and protein expression levels (mean ± S.E.M., n = 4, Mann–Whitney test). Densitometric analysis quantified REV-ERB in individual samples and was normalised to its own GAPDH loading control. HCV replication-dependent reporter activity was measured and expressed relative to control (shCtrl) cells (mean ± S.E.M., n = 6, Mann–Whitney test). b Anti-viral activity of SR9009 agonist is dependent on REV-ERB expression levels. sh Rev-eRbα and Ctrl HCV JFH-1 replicon cells described in ( a ) were treated with REV-ERB agonist SR9009 for 24 h, viral replication measured and the concentration of agonist required to inhibit viral replication by 50% defined (IC 50 ) (mean ± S.E.M., n = 3). c REV-ERBα overexpression inhibits HCV RNA replication. Huh-7 cells stably supporting a HCV JFH-1-LUC replicon were transfected with empty plasmid or vector expressing REV-ERBα and 48 h later protein expression assessed by western blotting and viral replication measured (mean ± S.E.M., n = 4, Mann–Whitney statistical test). Data are plotted relative to Ctrl untreated cells. d REV-ERB agonists cure HCV-infected cells. HCVcc SA13/JFH-1 infected Huh-7 cells were treated with increasing concentrations of REV-ERB agonists for 24 h and viral RNA or NS5A-expressing cells quantified and data expressed relative to Ctrl untreated cells. The experiment was performed in the presence of a neutralising anti-CD81 antibody to limit secondary rounds of infection (mean ± S.E.M., n = 3). e REV-ERB ligands inhibit the replication of diverse HCV genotypes. Huh-7 cells transiently supporting HCV sub-genomic replicons representing genotypes 1–3 were treated with the REV-ERB agonists SR9009 or GSK2667 and replication assessed 24 h later. The dose of agonist required to inhibit HCV RNA replication by 50% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)
Figure Legend Snippet: REV-ERBα inhibits hepatitis C virus (HCV) RNA replication. a Silencing Rev-erbα increases HCV replication. Huh-7 cells supporting a HCV JFH-1-LUC replicon were transduced with lentivirus encoding sh Rev-erbα or control and silencing confirmed by measuring Rev-erbα mRNA and protein expression levels (mean ± S.E.M., n = 4, Mann–Whitney test). Densitometric analysis quantified REV-ERB in individual samples and was normalised to its own GAPDH loading control. HCV replication-dependent reporter activity was measured and expressed relative to control (shCtrl) cells (mean ± S.E.M., n = 6, Mann–Whitney test). b Anti-viral activity of SR9009 agonist is dependent on REV-ERB expression levels. sh Rev-eRbα and Ctrl HCV JFH-1 replicon cells described in ( a ) were treated with REV-ERB agonist SR9009 for 24 h, viral replication measured and the concentration of agonist required to inhibit viral replication by 50% defined (IC 50 ) (mean ± S.E.M., n = 3). c REV-ERBα overexpression inhibits HCV RNA replication. Huh-7 cells stably supporting a HCV JFH-1-LUC replicon were transfected with empty plasmid or vector expressing REV-ERBα and 48 h later protein expression assessed by western blotting and viral replication measured (mean ± S.E.M., n = 4, Mann–Whitney statistical test). Data are plotted relative to Ctrl untreated cells. d REV-ERB agonists cure HCV-infected cells. HCVcc SA13/JFH-1 infected Huh-7 cells were treated with increasing concentrations of REV-ERB agonists for 24 h and viral RNA or NS5A-expressing cells quantified and data expressed relative to Ctrl untreated cells. The experiment was performed in the presence of a neutralising anti-CD81 antibody to limit secondary rounds of infection (mean ± S.E.M., n = 3). e REV-ERB ligands inhibit the replication of diverse HCV genotypes. Huh-7 cells transiently supporting HCV sub-genomic replicons representing genotypes 1–3 were treated with the REV-ERB agonists SR9009 or GSK2667 and replication assessed 24 h later. The dose of agonist required to inhibit HCV RNA replication by 50% (IC 50 ) was determined for all viral genotypes (mean ± S.E.M., n = 3)

Techniques Used: Transduction, Expressing, MANN-WHITNEY, Activity Assay, Concentration Assay, Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Infection

31) Product Images from "Exosome-mediated transfer of lncRNA-SNHG14 promotes trastuzumab chemoresistance in breast cancer"

Article Title: Exosome-mediated transfer of lncRNA-SNHG14 promotes trastuzumab chemoresistance in breast cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4467

Characterization of exosomes released from trastuzumab-resistant and -sensitive SKBR-3 cells. (A) Transmission electron microscopy images of the exosomes released by SKBR-3/Pr and SKBR-3/Tr cells. (B) Nanoparticle tracking analysis on an LM10 Nanosight unit demonstrating a mean size of 100 nm for SKBR-3/Tr and 120 nm for SKBR-3/Pr exosomes. The size distribution and relative concentration were calculated using the Nano-sight software. (C) Exosomal protein marker (CD63 and CD81) detection by western blotting from purified exosomes and cell extracts. (D) Flow cytometric analysis of the MFI for a panel of exosomal markers: CD9, CD63, CD81 and Alix. Data are presented as the median ± interquartile range of triplicate experiments. MFI, mean fluorescence intensity; CD, cluster of differentiation; Alix, programmed cell death 6-interacting protein.
Figure Legend Snippet: Characterization of exosomes released from trastuzumab-resistant and -sensitive SKBR-3 cells. (A) Transmission electron microscopy images of the exosomes released by SKBR-3/Pr and SKBR-3/Tr cells. (B) Nanoparticle tracking analysis on an LM10 Nanosight unit demonstrating a mean size of 100 nm for SKBR-3/Tr and 120 nm for SKBR-3/Pr exosomes. The size distribution and relative concentration were calculated using the Nano-sight software. (C) Exosomal protein marker (CD63 and CD81) detection by western blotting from purified exosomes and cell extracts. (D) Flow cytometric analysis of the MFI for a panel of exosomal markers: CD9, CD63, CD81 and Alix. Data are presented as the median ± interquartile range of triplicate experiments. MFI, mean fluorescence intensity; CD, cluster of differentiation; Alix, programmed cell death 6-interacting protein.

Techniques Used: Transmission Assay, Electron Microscopy, Concentration Assay, Software, Marker, Western Blot, Purification, Flow Cytometry, Fluorescence

32) Product Images from "Five-lncRNA signature in plasma exosomes serves as diagnostic biomarker for esophageal squamous cell carcinoma"

Article Title: Five-lncRNA signature in plasma exosomes serves as diagnostic biomarker for esophageal squamous cell carcinoma

Journal: Aging (Albany NY)

doi: 10.18632/aging.103559

Characterization of plasma exosomes from ESCC patients, esophagitis patients, and healthy controls. ( A ) Shape and structure of plasma exosomes isolated by ultracentrifugation under TEM. ( B ) Western blots of exosome membrane markers CD63, CD9, and CD81. ( C ) Sizes of plasma exosomes from ESCC patients, esophagitis patients, and healthy controls were analyzed by NTA.
Figure Legend Snippet: Characterization of plasma exosomes from ESCC patients, esophagitis patients, and healthy controls. ( A ) Shape and structure of plasma exosomes isolated by ultracentrifugation under TEM. ( B ) Western blots of exosome membrane markers CD63, CD9, and CD81. ( C ) Sizes of plasma exosomes from ESCC patients, esophagitis patients, and healthy controls were analyzed by NTA.

Techniques Used: Isolation, Transmission Electron Microscopy, Western Blot

33) Product Images from "Design of experiment (DoE)-driven in vitro and in vivo uptake studies of exosomes for pancreatic cancer delivery enabled by copper-free click chemistry-based labelling"

Article Title: Design of experiment (DoE)-driven in vitro and in vivo uptake studies of exosomes for pancreatic cancer delivery enabled by copper-free click chemistry-based labelling

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2020.1779458

Characterisation of PANC-1, B16-F10 and HEK-293 Exosomes (Exo). (a) Size distributions of PANC-1, B16-F10 and HEK-293 Exo obtained by Nanoparticle Tracking Analysis (NTA) using a NanoSight LM-10. The histograms indicate a similar size distribution profile for all the three exosomes. (b) Morphology characterisation by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Scale bar: 50 nm. (c) Detection of tetraspanins CD9, CD63 and CD81 on exosomes using flow cytometry. Exosomes were coupled to aldehyde/sulphate latex beads prior to detection. Exo-beads complex were subsequently stained using a 2-step labelling (anti-CD9, anti-CD63 or anti-CD81 1° Ab/Cy5-conjugated 2° Ab). Degree of expression of the markers are expressed as the fold increase in mean fluorescence intensity (MFI) values to Exo-beads complex stained with Cy5-conjugated 2° Ab, where an MFI ratio of 2 was set as the threshold for positive expression. (d) Detection of “do-not-eat-me” marker CD47 on PANC-1, B16-F10 and HEK-293 Exo. A MFI ratio of 2 was set as the threshold for positive expression. Values are expressed as mean ± SD ( n = 3). Statistical analysis were only conducted on human exosomes. * p
Figure Legend Snippet: Characterisation of PANC-1, B16-F10 and HEK-293 Exosomes (Exo). (a) Size distributions of PANC-1, B16-F10 and HEK-293 Exo obtained by Nanoparticle Tracking Analysis (NTA) using a NanoSight LM-10. The histograms indicate a similar size distribution profile for all the three exosomes. (b) Morphology characterisation by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Scale bar: 50 nm. (c) Detection of tetraspanins CD9, CD63 and CD81 on exosomes using flow cytometry. Exosomes were coupled to aldehyde/sulphate latex beads prior to detection. Exo-beads complex were subsequently stained using a 2-step labelling (anti-CD9, anti-CD63 or anti-CD81 1° Ab/Cy5-conjugated 2° Ab). Degree of expression of the markers are expressed as the fold increase in mean fluorescence intensity (MFI) values to Exo-beads complex stained with Cy5-conjugated 2° Ab, where an MFI ratio of 2 was set as the threshold for positive expression. (d) Detection of “do-not-eat-me” marker CD47 on PANC-1, B16-F10 and HEK-293 Exo. A MFI ratio of 2 was set as the threshold for positive expression. Values are expressed as mean ± SD ( n = 3). Statistical analysis were only conducted on human exosomes. * p

Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Flow Cytometry, Staining, Expressing, Fluorescence, Marker

34) Product Images from "Comparison and Investigation of Exosomes Derived from Platelet-Rich Plasma Activated by Different Agonists"

Article Title: Comparison and Investigation of Exosomes Derived from Platelet-Rich Plasma Activated by Different Agonists

Journal: Cell Transplantation

doi: 10.1177/09636897211017833

Western blot analysis of the surface biomarkers: CD81, TSG101, CD63, Flotillin, CD 9 on PRP-Exos; the source marker CD41; and the negative control (calnexin), which were found to be present in cell lysates. Densitometric analysis of western blot results: (A) representative western blot images and (B–E) densitometric analysis. Each lane was loaded with 60 µg of total exosomal protein. Statistical significance was determined using an independent-sample t-test. Data are shown as mean ± SD of values of three measurements in each group. n = 3 independent samples. N.S., not significant. * P
Figure Legend Snippet: Western blot analysis of the surface biomarkers: CD81, TSG101, CD63, Flotillin, CD 9 on PRP-Exos; the source marker CD41; and the negative control (calnexin), which were found to be present in cell lysates. Densitometric analysis of western blot results: (A) representative western blot images and (B–E) densitometric analysis. Each lane was loaded with 60 µg of total exosomal protein. Statistical significance was determined using an independent-sample t-test. Data are shown as mean ± SD of values of three measurements in each group. n = 3 independent samples. N.S., not significant. * P

Techniques Used: Western Blot, Marker, Negative Control

35) Product Images from "Exosomes Secreted from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Prevent Osteonecrosis of the Femoral Head by Promoting Angiogenesis"

Article Title: Exosomes Secreted from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Prevent Osteonecrosis of the Femoral Head by Promoting Angiogenesis

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.16951

Characterization of hiPS-MSCs and hiPS-MSC-Exos. (A) The fibroblast-like morphology of hiPS-MSCs shown in light microscopy images. (B) Surface markers of hiPS-MSCs analyzed by flow cytometry. (C) The morphology of hiPS-MSC-Exos shown by transmission electron microscopy. (D) Expression levels of CD9, CD63 and CD81 incorporation into hiPS-MSC-Exos shown by western blotting. (E) Identification of size and concentration of hiPS-MSC-Exos by nanoparticle analysis.
Figure Legend Snippet: Characterization of hiPS-MSCs and hiPS-MSC-Exos. (A) The fibroblast-like morphology of hiPS-MSCs shown in light microscopy images. (B) Surface markers of hiPS-MSCs analyzed by flow cytometry. (C) The morphology of hiPS-MSC-Exos shown by transmission electron microscopy. (D) Expression levels of CD9, CD63 and CD81 incorporation into hiPS-MSC-Exos shown by western blotting. (E) Identification of size and concentration of hiPS-MSC-Exos by nanoparticle analysis.

Techniques Used: Light Microscopy, Flow Cytometry, Transmission Assay, Electron Microscopy, Expressing, Western Blot, Concentration Assay

36) Product Images from "Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System"

Article Title: Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.553444

Characterization of MSC-EVs. (A) Representative AFM images of MSC-EVs obtained in the VWBR system, using MSC from three different human tissue sources (bone marrow, adipose tissue, and umbilical cord matrix). AFM height images (top) and respective 3D projections (bottom), capturing a total area of 10 × 10 μm. A close-up image focusing on a single EV is presented for each AFM height image. (B) Western blots of MSC lysates and MSC-EV samples. (i) Representative Western blot images of synthenin, CD63, CD81 and calnexin detection in MSC-EVs and corresponding WCL (i.e., cells) obtained from VWBR cultures. (ii) Western blot detection of synthenin, CD63 and CD81 in MSC-EV samples and corresponding WCL (i.e., cells), obtained from BM, AT and UCM MSC after EV production in static and VWBR systems. Detection of the housekeeping protein GAPDH in the same WCL preparations. (C) Zeta potential measurements of the surface charge of MSC-EVs (mV), obtained in either static or VWBR systems, using MSC from three different human sources (BM, AT, and UCM). Results correspond to one representative experiment for each condition. Results are presented as mean ± SD. AFM, atomic force microscopy; WCL, whole cell lysates; BM, bone marrow; AT, adipose tissue; UCM, umbilical cord matrix; VWBR, Vertical-Wheel TM bioreactor.
Figure Legend Snippet: Characterization of MSC-EVs. (A) Representative AFM images of MSC-EVs obtained in the VWBR system, using MSC from three different human tissue sources (bone marrow, adipose tissue, and umbilical cord matrix). AFM height images (top) and respective 3D projections (bottom), capturing a total area of 10 × 10 μm. A close-up image focusing on a single EV is presented for each AFM height image. (B) Western blots of MSC lysates and MSC-EV samples. (i) Representative Western blot images of synthenin, CD63, CD81 and calnexin detection in MSC-EVs and corresponding WCL (i.e., cells) obtained from VWBR cultures. (ii) Western blot detection of synthenin, CD63 and CD81 in MSC-EV samples and corresponding WCL (i.e., cells), obtained from BM, AT and UCM MSC after EV production in static and VWBR systems. Detection of the housekeeping protein GAPDH in the same WCL preparations. (C) Zeta potential measurements of the surface charge of MSC-EVs (mV), obtained in either static or VWBR systems, using MSC from three different human sources (BM, AT, and UCM). Results correspond to one representative experiment for each condition. Results are presented as mean ± SD. AFM, atomic force microscopy; WCL, whole cell lysates; BM, bone marrow; AT, adipose tissue; UCM, umbilical cord matrix; VWBR, Vertical-Wheel TM bioreactor.

Techniques Used: Western Blot, Microscopy

37) Product Images from "Exosomal miR-135a derived from human amnion mesenchymal stem cells promotes cutaneous wound healing in rats and fibroblast migration by directly inhibiting LATS2 expression"

Article Title: Exosomal miR-135a derived from human amnion mesenchymal stem cells promotes cutaneous wound healing in rats and fibroblast migration by directly inhibiting LATS2 expression

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-020-1570-9

Characterization of hAMSC-Exos. a TEM image of hAMSC-Exos ( n = 5). b Western blot analysis of exosomal markers CD9, CD63, and CD81 ( n = 3). c Size distribution of hAMSC-Exos (mean diameter = 103 nm, n = 3)
Figure Legend Snippet: Characterization of hAMSC-Exos. a TEM image of hAMSC-Exos ( n = 5). b Western blot analysis of exosomal markers CD9, CD63, and CD81 ( n = 3). c Size distribution of hAMSC-Exos (mean diameter = 103 nm, n = 3)

Techniques Used: Transmission Electron Microscopy, Western Blot

38) Product Images from "An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification"

Article Title: An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification

Journal: Theranostics

doi: 10.7150/thno.49047

Comparison of the apta-ELISA, apta-HCR-ELISA and apta-HCR-CRISPR assays. (A) apta-ELISA mechanism. In the apta-ELISA assay, EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in streptavidin-HRP. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (B) Detection of nucleolin + EVs by apta-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (C) apta-ELISA-HCR mechanism. EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in premixture HRP-labeled H1 and H2. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (D) Detection of nucleolin + EVs by apta-HCR-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (E) apta-HCR-CRISPR mechanism. Based on the apta-ELISA-HCR assay, the HCR products were targeted by Cas12a/crRNA duplex and triggered Cas12a to cleave the ssDNA-FQ reporter substrate, resulting in readable and accumulating FI proportional to the concentration of target positive EVs. (F) Detection of nucleolin + EVs by apta-HCR- CRISPR with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (G) Comparison of the LOD of apta-HCR-CRISPR, apta-HCR-ELISA and apta-ELISA in detecting nucleolin + EV spiked in PBS. (H) The concentration change in nucleolin + EVs is linearly related to the FI through fitting curves, Y= 7663 lg (EVs) - 12852 (R 2 = 0.9848). FI, fluorescence intensity. PBS served as a blank. The P values were calculated using a one-way ANOVA followed by a Sidak multiple-comparison with the former group. *, **, *** and **** represent P
Figure Legend Snippet: Comparison of the apta-ELISA, apta-HCR-ELISA and apta-HCR-CRISPR assays. (A) apta-ELISA mechanism. In the apta-ELISA assay, EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in streptavidin-HRP. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (B) Detection of nucleolin + EVs by apta-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (C) apta-ELISA-HCR mechanism. EVs were added to the anti-CD63, anti-CD81 and anti-CD9 MBs, incubated with a biotinylated aptamer, and washed and resuspended in premixture HRP-labeled H1 and H2. The reaction was launched by adding the substrate, and the OD was proportional to the original concentration of target positive EVs. (D) Detection of nucleolin + EVs by apta-HCR-ELISA with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (E) apta-HCR-CRISPR mechanism. Based on the apta-ELISA-HCR assay, the HCR products were targeted by Cas12a/crRNA duplex and triggered Cas12a to cleave the ssDNA-FQ reporter substrate, resulting in readable and accumulating FI proportional to the concentration of target positive EVs. (F) Detection of nucleolin + EVs by apta-HCR- CRISPR with serial concentrations of SUNE2 EVs spiked in PBS from 64-10 6 particles/µL. (G) Comparison of the LOD of apta-HCR-CRISPR, apta-HCR-ELISA and apta-ELISA in detecting nucleolin + EV spiked in PBS. (H) The concentration change in nucleolin + EVs is linearly related to the FI through fitting curves, Y= 7663 lg (EVs) - 12852 (R 2 = 0.9848). FI, fluorescence intensity. PBS served as a blank. The P values were calculated using a one-way ANOVA followed by a Sidak multiple-comparison with the former group. *, **, *** and **** represent P

Techniques Used: Enzyme-linked Immunosorbent Assay, CRISPR, Incubation, Concentration Assay, Labeling, Host-Cell Reactivation, Fluorescence

Schematic of apta-HCR-CRISPR. The EVs are captured by a cocktail of anti-CD63-, anti-CD81- and anti-CD9 antibody-coated beads and recognized with H0. The formed antibody-EV-H0 complexes trigger HCR and generate long repetitive target sequences that are specifically recognized by the added crRNA/Cas12a duplex. Target-activated Cas12a trans-cleaves nearby ssDNA-FQ reporter, resulting in readable and accumulating fluorescence signal proportional to the concentration of target positive EVs.
Figure Legend Snippet: Schematic of apta-HCR-CRISPR. The EVs are captured by a cocktail of anti-CD63-, anti-CD81- and anti-CD9 antibody-coated beads and recognized with H0. The formed antibody-EV-H0 complexes trigger HCR and generate long repetitive target sequences that are specifically recognized by the added crRNA/Cas12a duplex. Target-activated Cas12a trans-cleaves nearby ssDNA-FQ reporter, resulting in readable and accumulating fluorescence signal proportional to the concentration of target positive EVs.

Techniques Used: CRISPR, Fluorescence, Concentration Assay

39) Product Images from "CircHmbox1 Targeting miRNA-1247-5p Is Involved in the Regulation of Bone Metabolism by TNF-α in Postmenopausal Osteoporosis"

Article Title: CircHmbox1 Targeting miRNA-1247-5p Is Involved in the Regulation of Bone Metabolism by TNF-α in Postmenopausal Osteoporosis

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.594785

Exosomal circHmbox1 from osteoclasts treated with TNF-α inhibits osteoblast differentiation. (A) An electron microscopy image of exosomes. Scale bar = 0.5 μm. (B) The expressions of CD63, CD81 and HSP70 in cells lysates and exosomes secreted by RANKL-induced RAW 264.7 cells were analyzed by western blot. (C) The expressions of circHmbox1 were analyzed by qRT-PCR in osteoblasts incubated with exosomes from osteoclasts induced with RANKL and/or TNF-α. n = 4, ** P
Figure Legend Snippet: Exosomal circHmbox1 from osteoclasts treated with TNF-α inhibits osteoblast differentiation. (A) An electron microscopy image of exosomes. Scale bar = 0.5 μm. (B) The expressions of CD63, CD81 and HSP70 in cells lysates and exosomes secreted by RANKL-induced RAW 264.7 cells were analyzed by western blot. (C) The expressions of circHmbox1 were analyzed by qRT-PCR in osteoblasts incubated with exosomes from osteoclasts induced with RANKL and/or TNF-α. n = 4, ** P

Techniques Used: Electron Microscopy, Western Blot, Quantitative RT-PCR, Incubation

40) Product Images from "The Inflammatory Cytokine IL-3 Hampers Cardioprotection Mediated by Endothelial Cell-Derived Extracellular Vesicles Possibly via Their Protein Cargo"

Article Title: The Inflammatory Cytokine IL-3 Hampers Cardioprotection Mediated by Endothelial Cell-Derived Extracellular Vesicles Possibly via Their Protein Cargo

Journal: Cells

doi: 10.3390/cells10010013

Endothelial cell-derived extracellular vesicles (eEV) and eEV released in response to interleukin-3 (eEV-IL-3) characterization. ( A ) Representative images of NanoSight analyses and TEM performed on eEV and eEV-IL-3. ( B ) Western blot. Representative images of exosomal markers, CD29, CD63, and CD81, expressed in eEV and eEV-IL-3. GM130 protein expression served as a negative EV marker. Indeed, it was expressed only in the cell extract (Ctrl Human Umbilical Vein Cells (HUVEC)). ( C ) eEV and eEV-IL-3 characterization with MACSPlex Exosome Kit. ( D ) The table reports the expression of eEV and eEV-IL-3 exosomal and endothelial markers provided by mass spectrometry.
Figure Legend Snippet: Endothelial cell-derived extracellular vesicles (eEV) and eEV released in response to interleukin-3 (eEV-IL-3) characterization. ( A ) Representative images of NanoSight analyses and TEM performed on eEV and eEV-IL-3. ( B ) Western blot. Representative images of exosomal markers, CD29, CD63, and CD81, expressed in eEV and eEV-IL-3. GM130 protein expression served as a negative EV marker. Indeed, it was expressed only in the cell extract (Ctrl Human Umbilical Vein Cells (HUVEC)). ( C ) eEV and eEV-IL-3 characterization with MACSPlex Exosome Kit. ( D ) The table reports the expression of eEV and eEV-IL-3 exosomal and endothelial markers provided by mass spectrometry.

Techniques Used: Derivative Assay, Transmission Electron Microscopy, Western Blot, Expressing, Marker, Mass Spectrometry

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    Abcam anti cd8 antibody
    CD4 + TILs colocalize with PD-L1 + cells and contribute to poor survival. (a) Representative multi-IF images showing a sample with more CD4 + TILs than <t>CD8</t> + TILs in the stroma. Scale bar, 100 μ m. (b) Representative multi-IF images showing a sample with more CD8 + TILs than CD4 + TILs in the stroma. Scale bar, 100 μ m. (c) A pie chart was plotted according to IHC staining results showing 84% of patients have more CD4 + TILs than CD8 + TILs in the stroma. (d) Representative multi-IF images showing a sample with PD-L1 + cells colocalizing more with CD4 + TILs than with CD8 + TILs in the stroma. Scale bar, 100 μ m. (e) The Kaplan–Meier survival analysis showing patients with elevated levels of CD4 + TILs (red line, n = 26) have poor OS, compared to patients with low levels of CD4 + TILs (blue line, n = 51). (f) The Kaplan–Meier survival analysis showing patients with a low CD8/CD4 ratio (blue line, n = 46) have poor OS, compared to patients with a high CD8/CD4 ratio (red line, n = 31).
    Anti Cd8 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd81  (Abcam)
    99
    Abcam cd81
    Gene expression analysis of CD9 and <t>CD81</t> in prostate cancer compared to normal samples in publicly available gene expression profiling datasets. (A–D): Gene expression boxplots for CD9. (A) TCGA dataset-prostate adenocarcinoma versus normal. (B) Wallace dataset- prostate adenocarcinoma vs. normal. (C) Lapointe dataset- prostate carcinoma vs. normal. (D) Yu dataset-prostate carcinoma vs. normal. ( E -H): Gene expression boxplots for CD81. (E) TCGA dataset-prostate adenocarcinoma vs. normal. (F) Vanaja dataset-prostate adenocarcinoma vs. normal. (G) Taylor dataset-prostate carcinoma vs. normal. (H) Singh dataset- prostate carcinoma vs. normal. 1, Normal; 2, Carcinoma or Adenocarcinoma.
    Cd81, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rat anti mouse cd8 monoclonal antibody
    A : Fluorescence microscopy revealed lower protein expression of B2M in MN-siB2M–treated grafts compared with control probe grafts 2 weeks after adoptive transfer (green, insulin; red, B2M; blue, DAPI nuclear stain; magnification bar = 50 μm). B : Fluorescence microscopy showed markedly lower <t>CD8</t> + T-cell infiltration in MN-siB2M–treated grafts compared with control islet grafts 2 weeks after adoptive transfer (green, insulin; red, CD8; blue, DAPI nuclear stain; magnification bar = 40 μm). C : Western blot analysis confirmed notably lower B2M protein expression in MN-siB2M–treated islet grafts compared with control grafts. (A high-quality digital representation of this figure is available in the online issue.)
    Rat Anti Mouse Cd8 Monoclonal Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti cd81
    Immunolocalization of exosome markers in the RPE/choroid complex. (A): CD63 (red); AMD eye from a 74-year-old male. Anti-CD63 antibody labels large amorphous areas (CD63: arrows) in drusen. <t>(B):CD81</t> (red); AMD eye from a 96-year-old male (CD81, arrows). (C): LAMP2 (red). AMD eye from a 93-year-old (LAMP2, arrow). (D): CD63 (red); Non-AMD eye from a 75-year-old male as an age-matched control. No CD63 labeling was seen in the drusen from any age-matched control (n = 10). (E–H): CD63 (green) co-localization with proteins that are known to be in drusen. (E) amyloid β (red) showed no co-localization with CD63 (arrow). (F) α B crystalline (red) showed co-localization with CD63 (arrowhead). (G) C5b-9 (red) showed no co-localization with CD63 (arrow). (H) CFH (red) showed co-localization with CD63 (arrowhead). Dr, drusen. Blue: DAPI.
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    CD4 + TILs colocalize with PD-L1 + cells and contribute to poor survival. (a) Representative multi-IF images showing a sample with more CD4 + TILs than CD8 + TILs in the stroma. Scale bar, 100 μ m. (b) Representative multi-IF images showing a sample with more CD8 + TILs than CD4 + TILs in the stroma. Scale bar, 100 μ m. (c) A pie chart was plotted according to IHC staining results showing 84% of patients have more CD4 + TILs than CD8 + TILs in the stroma. (d) Representative multi-IF images showing a sample with PD-L1 + cells colocalizing more with CD4 + TILs than with CD8 + TILs in the stroma. Scale bar, 100 μ m. (e) The Kaplan–Meier survival analysis showing patients with elevated levels of CD4 + TILs (red line, n = 26) have poor OS, compared to patients with low levels of CD4 + TILs (blue line, n = 51). (f) The Kaplan–Meier survival analysis showing patients with a low CD8/CD4 ratio (blue line, n = 46) have poor OS, compared to patients with a high CD8/CD4 ratio (red line, n = 31).

    Journal: Journal of Immunology Research

    Article Title: Tumor-Infiltrating Immune Cells and PD-L1 as Prognostic Biomarkers in Primary Esophageal Small Cell Carcinoma

    doi: 10.1155/2020/8884683

    Figure Lengend Snippet: CD4 + TILs colocalize with PD-L1 + cells and contribute to poor survival. (a) Representative multi-IF images showing a sample with more CD4 + TILs than CD8 + TILs in the stroma. Scale bar, 100 μ m. (b) Representative multi-IF images showing a sample with more CD8 + TILs than CD4 + TILs in the stroma. Scale bar, 100 μ m. (c) A pie chart was plotted according to IHC staining results showing 84% of patients have more CD4 + TILs than CD8 + TILs in the stroma. (d) Representative multi-IF images showing a sample with PD-L1 + cells colocalizing more with CD4 + TILs than with CD8 + TILs in the stroma. Scale bar, 100 μ m. (e) The Kaplan–Meier survival analysis showing patients with elevated levels of CD4 + TILs (red line, n = 26) have poor OS, compared to patients with low levels of CD4 + TILs (blue line, n = 51). (f) The Kaplan–Meier survival analysis showing patients with a low CD8/CD4 ratio (blue line, n = 46) have poor OS, compared to patients with a high CD8/CD4 ratio (red line, n = 31).

    Article Snippet: Immunohistochemistry StainingIHC staining was performed using anti-PD-L1 antibody (1 : 500) (clone 28-8, Abcam, Cambridge, UK) [ – ], anti-CD4 antibody (1 : 500) (clone EPR6855, Abcam, Cambridge, UK), anti-CD8 antibody (1 : 200) (rabbit polyclonal anti-CD8, Abcam, Cambridge, UK), anti-CD163 antibody (1 : 500) (clone EPR19518, Abcam, Cambridge, UK), and anti-FoxP3 antibody (1 : 100) (clone 236A/E7, Abcam, Cambridge, UK) as the primary antibodies.

    Techniques: Immunohistochemistry, Staining

    Expression and prognostic value of FoxP3 in PESCC. (a) Representative multi-IF images of two samples showing FoxP3/CD4 ratios in the stroma. Scale bar, 100 μ m. (b) Representative multi-IF images showing two samples with high (left) and low (right) FoxP3/CD8 ratios in stroma. Scale bar, 100 μ m. (c) Rich FoxP3 (red line, n = 26) was associated with significantly shorter OS than poor FoxP3 (blue line, n = 51). (d) A high FoxP3/CD8 ratio (red line, n = 28) was associated with shorter OS than a low FoxP3/CD8 ratio (blue line, n = 49).

    Journal: Journal of Immunology Research

    Article Title: Tumor-Infiltrating Immune Cells and PD-L1 as Prognostic Biomarkers in Primary Esophageal Small Cell Carcinoma

    doi: 10.1155/2020/8884683

    Figure Lengend Snippet: Expression and prognostic value of FoxP3 in PESCC. (a) Representative multi-IF images of two samples showing FoxP3/CD4 ratios in the stroma. Scale bar, 100 μ m. (b) Representative multi-IF images showing two samples with high (left) and low (right) FoxP3/CD8 ratios in stroma. Scale bar, 100 μ m. (c) Rich FoxP3 (red line, n = 26) was associated with significantly shorter OS than poor FoxP3 (blue line, n = 51). (d) A high FoxP3/CD8 ratio (red line, n = 28) was associated with shorter OS than a low FoxP3/CD8 ratio (blue line, n = 49).

    Article Snippet: Immunohistochemistry StainingIHC staining was performed using anti-PD-L1 antibody (1 : 500) (clone 28-8, Abcam, Cambridge, UK) [ – ], anti-CD4 antibody (1 : 500) (clone EPR6855, Abcam, Cambridge, UK), anti-CD8 antibody (1 : 200) (rabbit polyclonal anti-CD8, Abcam, Cambridge, UK), anti-CD163 antibody (1 : 500) (clone EPR19518, Abcam, Cambridge, UK), and anti-FoxP3 antibody (1 : 100) (clone 236A/E7, Abcam, Cambridge, UK) as the primary antibodies.

    Techniques: Expressing

    The expression of PD-L1 in the stroma correlates with tumor-infiltrating immune cells in PESCC. (a) Representative immunohistochemistry (IHC) images showing different expression patterns of PD-L1 in PESCC tissues. Scale bar, 100 μ m. S: stroma; T: tumor. (b) Distribution of different expression patterns of PD-L1 are plotted in the pie chart. (c) The Kaplan–Meier survival analysis of overall survival (OS) in a cohort of 77 PESCC patients according to positive (red line, n = 26) and negative (blue line, n = 51) PD-L1 expression. (d) Representative IHC images showing PD-L1, CD4, CD8, and CD163 expression in the stroma using consecutive sections of PD-L1-positive and PD-L1-negative specimens. Scale bar, 100 μ m. S: stroma; T: tumor. (e) The presence of PD-L1 is positively associated with the number of CD4 + TILs, CD8 + TILs, and CD163 + TAMs ( ∗∗ p

    Journal: Journal of Immunology Research

    Article Title: Tumor-Infiltrating Immune Cells and PD-L1 as Prognostic Biomarkers in Primary Esophageal Small Cell Carcinoma

    doi: 10.1155/2020/8884683

    Figure Lengend Snippet: The expression of PD-L1 in the stroma correlates with tumor-infiltrating immune cells in PESCC. (a) Representative immunohistochemistry (IHC) images showing different expression patterns of PD-L1 in PESCC tissues. Scale bar, 100 μ m. S: stroma; T: tumor. (b) Distribution of different expression patterns of PD-L1 are plotted in the pie chart. (c) The Kaplan–Meier survival analysis of overall survival (OS) in a cohort of 77 PESCC patients according to positive (red line, n = 26) and negative (blue line, n = 51) PD-L1 expression. (d) Representative IHC images showing PD-L1, CD4, CD8, and CD163 expression in the stroma using consecutive sections of PD-L1-positive and PD-L1-negative specimens. Scale bar, 100 μ m. S: stroma; T: tumor. (e) The presence of PD-L1 is positively associated with the number of CD4 + TILs, CD8 + TILs, and CD163 + TAMs ( ∗∗ p

    Article Snippet: Immunohistochemistry StainingIHC staining was performed using anti-PD-L1 antibody (1 : 500) (clone 28-8, Abcam, Cambridge, UK) [ – ], anti-CD4 antibody (1 : 500) (clone EPR6855, Abcam, Cambridge, UK), anti-CD8 antibody (1 : 200) (rabbit polyclonal anti-CD8, Abcam, Cambridge, UK), anti-CD163 antibody (1 : 500) (clone EPR19518, Abcam, Cambridge, UK), and anti-FoxP3 antibody (1 : 100) (clone 236A/E7, Abcam, Cambridge, UK) as the primary antibodies.

    Techniques: Expressing, Immunohistochemistry

    CD4 + TILs colocalize with PD-L1 + CD163 + TAMs. Representative multicolor immunofluorescence (multi-IF) images showing (a) high and (b) low colocalization of PD-L1 + cells with CD163 + TAMs. Scale bar, 100 μ m. Representative multi-IF images showing costaining of (c) CD4 + PD-L1 + CD163 + TILs and (d) CD8 + PD-L1 + CD163 + TILs in serial sections of the same specimen. Cells were counterstained with DAPI (blue, nucleus). Scale bar, 100 μ m.

    Journal: Journal of Immunology Research

    Article Title: Tumor-Infiltrating Immune Cells and PD-L1 as Prognostic Biomarkers in Primary Esophageal Small Cell Carcinoma

    doi: 10.1155/2020/8884683

    Figure Lengend Snippet: CD4 + TILs colocalize with PD-L1 + CD163 + TAMs. Representative multicolor immunofluorescence (multi-IF) images showing (a) high and (b) low colocalization of PD-L1 + cells with CD163 + TAMs. Scale bar, 100 μ m. Representative multi-IF images showing costaining of (c) CD4 + PD-L1 + CD163 + TILs and (d) CD8 + PD-L1 + CD163 + TILs in serial sections of the same specimen. Cells were counterstained with DAPI (blue, nucleus). Scale bar, 100 μ m.

    Article Snippet: Immunohistochemistry StainingIHC staining was performed using anti-PD-L1 antibody (1 : 500) (clone 28-8, Abcam, Cambridge, UK) [ – ], anti-CD4 antibody (1 : 500) (clone EPR6855, Abcam, Cambridge, UK), anti-CD8 antibody (1 : 200) (rabbit polyclonal anti-CD8, Abcam, Cambridge, UK), anti-CD163 antibody (1 : 500) (clone EPR19518, Abcam, Cambridge, UK), and anti-FoxP3 antibody (1 : 100) (clone 236A/E7, Abcam, Cambridge, UK) as the primary antibodies.

    Techniques: Immunofluorescence

    Gene expression analysis of CD9 and CD81 in prostate cancer compared to normal samples in publicly available gene expression profiling datasets. (A–D): Gene expression boxplots for CD9. (A) TCGA dataset-prostate adenocarcinoma versus normal. (B) Wallace dataset- prostate adenocarcinoma vs. normal. (C) Lapointe dataset- prostate carcinoma vs. normal. (D) Yu dataset-prostate carcinoma vs. normal. ( E -H): Gene expression boxplots for CD81. (E) TCGA dataset-prostate adenocarcinoma vs. normal. (F) Vanaja dataset-prostate adenocarcinoma vs. normal. (G) Taylor dataset-prostate carcinoma vs. normal. (H) Singh dataset- prostate carcinoma vs. normal. 1, Normal; 2, Carcinoma or Adenocarcinoma.

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

    doi: 10.1016/j.matbio.2018.08.004

    Figure Lengend Snippet: Gene expression analysis of CD9 and CD81 in prostate cancer compared to normal samples in publicly available gene expression profiling datasets. (A–D): Gene expression boxplots for CD9. (A) TCGA dataset-prostate adenocarcinoma versus normal. (B) Wallace dataset- prostate adenocarcinoma vs. normal. (C) Lapointe dataset- prostate carcinoma vs. normal. (D) Yu dataset-prostate carcinoma vs. normal. ( E -H): Gene expression boxplots for CD81. (E) TCGA dataset-prostate adenocarcinoma vs. normal. (F) Vanaja dataset-prostate adenocarcinoma vs. normal. (G) Taylor dataset-prostate carcinoma vs. normal. (H) Singh dataset- prostate carcinoma vs. normal. 1, Normal; 2, Carcinoma or Adenocarcinoma.

    Article Snippet: The following antibodies (Abs) were used for immunoblotting (IB) analysis: mouse monoclonal Abs to PSMA (ab-19,071; Abcam), CD9 (sc-13,118; Santa Cruz Biotechnology), CD81 (ab-23,505; Abcam), CD63 (ab-8219; Abcam), GM130 (61,820; BD Biosciences), EpCAM (2929; Cell Signaling Technology), TUBULIN (T-8535; Sigma); rabbit mAb to TSG101 (ab-125,011; Abcam); rabbit polyclonal Abs (pAbs) to FLOTILLIN1 (FLOT1) (ab-41,927; Abcam), GFP (ab-6556; Abcam), CALNEXIN (CANX) (sc-11,397; Santa Cruz Biotechnology), ERK1 (sc-93; Santa Cruz Biotechnology), and Rb-IgG (Sigma).

    Techniques: Expressing

    GFP-tagged αvβ3 integrin is expressed in prostate cancer cell ExVs, and it is localized in distant lesions in vivo. (A) IB analysis of expression of GFP in total cell lysates (TCL) from parental C4-2B, C4-2B-GFP (Mock) and C4-2B-β3-GFP cells. Calnexin (CANX) was used as loading control. (B) IB analysis of expression of αv, β3, CD63, CD81 (non-reducing conditions) and GFP, β3, TSG101, CD9 (reducing conditions) in lysates from ExVs derived from C4-2B-β3-GFP cells by differential ultracentrifugation. (C) In vivo fluorescence and corresponding phase contrast images are shown for liver, DU145 tumor, and prostate isolated from NSG mice that were injected subcutaneously with C4-2B-β3-GFP cells on the right side and non-transfected DU145 cells on the left side. Two representative samples are shown. (D) NTA for size distribution and concentration of GFP-positive ExVs isolated by differential ultracentrifugation from plasma of the mice subcutaneously injected with C4-2B-β3-GFP cells. One representative sample is shown.

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

    doi: 10.1016/j.matbio.2018.08.004

    Figure Lengend Snippet: GFP-tagged αvβ3 integrin is expressed in prostate cancer cell ExVs, and it is localized in distant lesions in vivo. (A) IB analysis of expression of GFP in total cell lysates (TCL) from parental C4-2B, C4-2B-GFP (Mock) and C4-2B-β3-GFP cells. Calnexin (CANX) was used as loading control. (B) IB analysis of expression of αv, β3, CD63, CD81 (non-reducing conditions) and GFP, β3, TSG101, CD9 (reducing conditions) in lysates from ExVs derived from C4-2B-β3-GFP cells by differential ultracentrifugation. (C) In vivo fluorescence and corresponding phase contrast images are shown for liver, DU145 tumor, and prostate isolated from NSG mice that were injected subcutaneously with C4-2B-β3-GFP cells on the right side and non-transfected DU145 cells on the left side. Two representative samples are shown. (D) NTA for size distribution and concentration of GFP-positive ExVs isolated by differential ultracentrifugation from plasma of the mice subcutaneously injected with C4-2B-β3-GFP cells. One representative sample is shown.

    Article Snippet: The following antibodies (Abs) were used for immunoblotting (IB) analysis: mouse monoclonal Abs to PSMA (ab-19,071; Abcam), CD9 (sc-13,118; Santa Cruz Biotechnology), CD81 (ab-23,505; Abcam), CD63 (ab-8219; Abcam), GM130 (61,820; BD Biosciences), EpCAM (2929; Cell Signaling Technology), TUBULIN (T-8535; Sigma); rabbit mAb to TSG101 (ab-125,011; Abcam); rabbit polyclonal Abs (pAbs) to FLOTILLIN1 (FLOT1) (ab-41,927; Abcam), GFP (ab-6556; Abcam), CALNEXIN (CANX) (sc-11,397; Santa Cruz Biotechnology), ERK1 (sc-93; Santa Cruz Biotechnology), and Rb-IgG (Sigma).

    Techniques: In Vivo, Expressing, Derivative Assay, Fluorescence, Isolation, Mouse Assay, Injection, Transfection, Concentration Assay

    Size distribution analysis and differences in β3 integrin levels in ExVs from plasma of prostate cancer patients compared to subjects not affected by cancer. (A) NTA of ExVs isolated by differential ultracentrifugation from plasma of individuals not affected by cancer (left panel) and prostate cancer patients (right panel). (B) IB analysis of β3, CD9, CD81, and αv levels in ExVs isolated by differential ultracentrifugation from plasma of prostate cancer patients compared to age-matched individuals not affected by cancer. CANX was analyzed as a marker absent in exosomes. Lanes 1, 2, and 3: EV lysates isolated after the plasma was pooled from at least two subjects not affected by cancer (total of 7 biological samples represented in 3 lanes); lanes 4–8: EV lysates from individual patients. 30 μg of exosome lysates were loaded in each lane. (C) lodixanol gradient purified Exosomes (Exo) from prostate cancer patient plasma (pooled from n = 3) were immunocaptured with an antibody to Prostate Specific Membrane Antigen (PSMA) or isotype rabbit immunoglobulin (Rb-IgG) conjugated with Dynabeads M-270 epoxy magnetic beads, according to the manufacturer’s protocol. The immunocaptured whole exosomes were lysed with RIPA buffer, and lysates were separated by SDS-PAGE (7.5% gel). IB analysis shows expression of β3, CD9 and CD63 (exosomal markers), Trop-2, and PSMA; in contrast, TSG101 (exosomal marker), CANX and EpCAM were not detected. HC-IgG, heavy chain IgG.

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

    doi: 10.1016/j.matbio.2018.08.004

    Figure Lengend Snippet: Size distribution analysis and differences in β3 integrin levels in ExVs from plasma of prostate cancer patients compared to subjects not affected by cancer. (A) NTA of ExVs isolated by differential ultracentrifugation from plasma of individuals not affected by cancer (left panel) and prostate cancer patients (right panel). (B) IB analysis of β3, CD9, CD81, and αv levels in ExVs isolated by differential ultracentrifugation from plasma of prostate cancer patients compared to age-matched individuals not affected by cancer. CANX was analyzed as a marker absent in exosomes. Lanes 1, 2, and 3: EV lysates isolated after the plasma was pooled from at least two subjects not affected by cancer (total of 7 biological samples represented in 3 lanes); lanes 4–8: EV lysates from individual patients. 30 μg of exosome lysates were loaded in each lane. (C) lodixanol gradient purified Exosomes (Exo) from prostate cancer patient plasma (pooled from n = 3) were immunocaptured with an antibody to Prostate Specific Membrane Antigen (PSMA) or isotype rabbit immunoglobulin (Rb-IgG) conjugated with Dynabeads M-270 epoxy magnetic beads, according to the manufacturer’s protocol. The immunocaptured whole exosomes were lysed with RIPA buffer, and lysates were separated by SDS-PAGE (7.5% gel). IB analysis shows expression of β3, CD9 and CD63 (exosomal markers), Trop-2, and PSMA; in contrast, TSG101 (exosomal marker), CANX and EpCAM were not detected. HC-IgG, heavy chain IgG.

    Article Snippet: The following antibodies (Abs) were used for immunoblotting (IB) analysis: mouse monoclonal Abs to PSMA (ab-19,071; Abcam), CD9 (sc-13,118; Santa Cruz Biotechnology), CD81 (ab-23,505; Abcam), CD63 (ab-8219; Abcam), GM130 (61,820; BD Biosciences), EpCAM (2929; Cell Signaling Technology), TUBULIN (T-8535; Sigma); rabbit mAb to TSG101 (ab-125,011; Abcam); rabbit polyclonal Abs (pAbs) to FLOTILLIN1 (FLOT1) (ab-41,927; Abcam), GFP (ab-6556; Abcam), CALNEXIN (CANX) (sc-11,397; Santa Cruz Biotechnology), ERK1 (sc-93; Santa Cruz Biotechnology), and Rb-IgG (Sigma).

    Techniques: Isolation, Marker, Purification, Magnetic Beads, SDS Page, Expressing

    A : Fluorescence microscopy revealed lower protein expression of B2M in MN-siB2M–treated grafts compared with control probe grafts 2 weeks after adoptive transfer (green, insulin; red, B2M; blue, DAPI nuclear stain; magnification bar = 50 μm). B : Fluorescence microscopy showed markedly lower CD8 + T-cell infiltration in MN-siB2M–treated grafts compared with control islet grafts 2 weeks after adoptive transfer (green, insulin; red, CD8; blue, DAPI nuclear stain; magnification bar = 40 μm). C : Western blot analysis confirmed notably lower B2M protein expression in MN-siB2M–treated islet grafts compared with control grafts. (A high-quality digital representation of this figure is available in the online issue.)

    Journal: Diabetes

    Article Title: A Theranostic Small Interfering RNA Nanoprobe Protects Pancreatic Islet Grafts From Adoptively Transferred Immune Rejection

    doi: 10.2337/db12-0441

    Figure Lengend Snippet: A : Fluorescence microscopy revealed lower protein expression of B2M in MN-siB2M–treated grafts compared with control probe grafts 2 weeks after adoptive transfer (green, insulin; red, B2M; blue, DAPI nuclear stain; magnification bar = 50 μm). B : Fluorescence microscopy showed markedly lower CD8 + T-cell infiltration in MN-siB2M–treated grafts compared with control islet grafts 2 weeks after adoptive transfer (green, insulin; red, CD8; blue, DAPI nuclear stain; magnification bar = 40 μm). C : Western blot analysis confirmed notably lower B2M protein expression in MN-siB2M–treated islet grafts compared with control grafts. (A high-quality digital representation of this figure is available in the online issue.)

    Article Snippet: For human insulin and mouse CD8+ T cells double-staining, sections were incubated with a guinea pig anti-human insulin primary antibody (1:200 dilution, Abcam) and rat anti-mouse CD8 monoclonal antibody (1:50 dilution, Abcam), followed by an FITC-labeled goat anti-guinea pig secondary IgG (1:100 dilution, Abcam) and Alexa Fluor 594 conjugated secondary goat anti-rat IgG (1:50 dilution, Invitrogen).

    Techniques: Fluorescence, Microscopy, Expressing, Adoptive Transfer Assay, Staining, Western Blot

    Immunolocalization of exosome markers in the RPE/choroid complex. (A): CD63 (red); AMD eye from a 74-year-old male. Anti-CD63 antibody labels large amorphous areas (CD63: arrows) in drusen. (B):CD81 (red); AMD eye from a 96-year-old male (CD81, arrows). (C): LAMP2 (red). AMD eye from a 93-year-old (LAMP2, arrow). (D): CD63 (red); Non-AMD eye from a 75-year-old male as an age-matched control. No CD63 labeling was seen in the drusen from any age-matched control (n = 10). (E–H): CD63 (green) co-localization with proteins that are known to be in drusen. (E) amyloid β (red) showed no co-localization with CD63 (arrow). (F) α B crystalline (red) showed co-localization with CD63 (arrowhead). (G) C5b-9 (red) showed no co-localization with CD63 (arrow). (H) CFH (red) showed co-localization with CD63 (arrowhead). Dr, drusen. Blue: DAPI.

    Journal: PLoS ONE

    Article Title: Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration

    doi: 10.1371/journal.pone.0004160

    Figure Lengend Snippet: Immunolocalization of exosome markers in the RPE/choroid complex. (A): CD63 (red); AMD eye from a 74-year-old male. Anti-CD63 antibody labels large amorphous areas (CD63: arrows) in drusen. (B):CD81 (red); AMD eye from a 96-year-old male (CD81, arrows). (C): LAMP2 (red). AMD eye from a 93-year-old (LAMP2, arrow). (D): CD63 (red); Non-AMD eye from a 75-year-old male as an age-matched control. No CD63 labeling was seen in the drusen from any age-matched control (n = 10). (E–H): CD63 (green) co-localization with proteins that are known to be in drusen. (E) amyloid β (red) showed no co-localization with CD63 (arrow). (F) α B crystalline (red) showed co-localization with CD63 (arrowhead). (G) C5b-9 (red) showed no co-localization with CD63 (arrow). (H) CFH (red) showed co-localization with CD63 (arrowhead). Dr, drusen. Blue: DAPI.

    Article Snippet: 110 µl of 1 M glycine was added (i.e., 100 mM final), mixed gently and let stand on the bench at room temperature for 30 min. Beads were re-suspended in 0.5 ml PBS/0.5% BSA, 10 µl coated beads were incubated with 50 µl primary antibodies: mouse anti-CFH (1∶50, Serotec), mouse anti-CD63 (1∶50, Abcam), mouse ant-LAMP2 (1∶50, Abcam), mouse anti-CD81 (1∶50, Abcam), mouse anti-C3 (1∶50, Abcam).

    Techniques: Labeling

    Analyses of exosomes by FACS. (A) Exoxomes are CD63, LAMP2, CD81 and C3 positive, but CFH negative. Isotype control: Black; CFH: light green; CD63: blue; LAMP2: red; CD81: dark green; C3: magenta. (B) Added CFH bound to exosomes in a dose-dependent manner. (C) Added CFH did not alter the amount of C3 on exosomes. For B and C: CFH 0.5 mg/mL: black; CFH 0.05 mg/mL: blue; CFH 0.005 mg/mL: purple; CFH 0.0005 mg/mL: green; CFH 0 mg/mL: red.

    Journal: PLoS ONE

    Article Title: Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration

    doi: 10.1371/journal.pone.0004160

    Figure Lengend Snippet: Analyses of exosomes by FACS. (A) Exoxomes are CD63, LAMP2, CD81 and C3 positive, but CFH negative. Isotype control: Black; CFH: light green; CD63: blue; LAMP2: red; CD81: dark green; C3: magenta. (B) Added CFH bound to exosomes in a dose-dependent manner. (C) Added CFH did not alter the amount of C3 on exosomes. For B and C: CFH 0.5 mg/mL: black; CFH 0.05 mg/mL: blue; CFH 0.005 mg/mL: purple; CFH 0.0005 mg/mL: green; CFH 0 mg/mL: red.

    Article Snippet: 110 µl of 1 M glycine was added (i.e., 100 mM final), mixed gently and let stand on the bench at room temperature for 30 min. Beads were re-suspended in 0.5 ml PBS/0.5% BSA, 10 µl coated beads were incubated with 50 µl primary antibodies: mouse anti-CFH (1∶50, Serotec), mouse anti-CD63 (1∶50, Abcam), mouse ant-LAMP2 (1∶50, Abcam), mouse anti-CD81 (1∶50, Abcam), mouse anti-C3 (1∶50, Abcam).

    Techniques: FACS