Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery

doi: 10.1080/20013078.2020.1747206

Figure Lengend Snippet: Characterization of the IEV protein cargo for EV markers. (a and b) Commonly EV-associated proteins Hsp70, TSG101, CD9 and CD81 along with GM130 and α-tubulin were analysed by western blotting from the pooled fractions 1–6 of cEVs and IEVs or cell lysate (CL) of PC-3 (a) and A549 (b) cells. Also fractions 7–10 from IEV samples were analysed. Representative blots are shown with three independent samples of IEVs/cEVs and two independent samples of CL and fractions 7–10. While other protein markers seemed to be depleted from the IEVs and cEVs, membrane proteins CD9 and CD81 were enriched in those samples. (c and d) the samples were also analysed by stain-free SDS-PAGE, showing a distinct pattern of proteins in both pools of fractions 1–6 and 7–10 when compared to CL.

Article Snippet: Each strip was then incubated with primary antibodies diluted in 5% BSA-TBS-T (mouse anti-TSG101 (51/TSG101, 1:500, reducing conditions, BD Biosciences, Frankling Lakes, NJ, USA), mouse anti-Hsp70 (7/Hsp70, 1:500, reducing conditions, BD Biosciences), mouse anti-CD9 (HBM-CD9, 1:1000, non-reducing conditions, HansaBioMed Life Sciences, Tallinn, Estonia), mouse anti-CD81 (HBM-CD81-EM4, 1:500, non-reducing conditions, HansaBioMed Life Sciences), mouse anti-α-tubulin (DM1A, 1:500, reducing conditions, Sigma Aldrich), rabbit anti-GM130 (NBP1-89756, 1:250, reducing conditions, Novus Biologicals, Centennial, Colorado, USA), rabbit anti-Adenovirus type 5 antibody (ab6982, 1:4000, non-reducing conditions, Abcam)) overnight at +4°C.

Techniques: Western Blot, Staining, SDS Page

Journal: Journal of Extracellular Vesicles

Article Title: Identification of specific markers for human pluripotent stem cell‐derived small extracellular vesicles

doi: 10.1002/jev2.12409

Figure Lengend Snippet: Characterization of PSC‐sEVs. The morphology, particle size, classical protein markers, yields in terms of particles and proteins, and surface potential of PSC‐sEVs were detected. (a) Representative TEM images of ESC‐sEVs and iPSC‐sEVs. Scale bar, 200nm. (b) Representative profiles of size distribution of ESC‐sEVs and iPSC‐sEVs determined by NanoFCM. (c) Representative bands of Western Blot for sEVs markers (CD9, CD63, Alix, and TSG101) and non‐sEVs markers (Calnexin and GM130) in PSCs and PSC‐sEVs. (d‐f) Evaluation of ESC‐sEVs and iPSC‐sEVs yield in terms of the particle concentration and the protein concentration (n=5). (g) Zeta potential of ESC‐sEVs and iPSC‐sEVs (n=10). PSC‐sEVs, pluripotent stem cell‐derived sEVs; TEM, transmission electron microscope; ESC‐sEVs, embryonic stem cell‐derived sEVs; iPSC‐sEVs, induced pluripotent stem cell‐derived sEVs.

Article Snippet: The following antibodies were used for immunofluorescent staining: Alexa Fluor 488 (AF488) Mouse Anti‐Human CD9 (NanoFCM, NHA009‐A488‐50T, 1:10), AF488 Mouse Anti‐Human CD63 (NanoFCM, NHA063‐A488‐50T, 1:10), FITC Mouse Anti‐Human CD81 (NanoFCM, NHA‐FITC‐50T, 1:10), AF488 anti‐human PODXL Antibody (Abcam, ab208254, 1:20), AF488 anti‐human SSEA‐4 Antibody (BioLegend, 330411, 1:20), AF488 anti‐human TRA‐1‐81 Antibody (BioLegend, 330709, 1:20), AF488 anti‐human TRA‐1‐60‐R Antibody (BioLegend, 330613, 1:20).

Techniques: Western Blot, Concentration Assay, Protein Concentration, Zeta Potential Analyzer, Derivative Assay, Transmission Assay, Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Identification of specific markers for human pluripotent stem cell‐derived small extracellular vesicles

doi: 10.1002/jev2.12409

Figure Lengend Snippet: Evaluation of surface markers of PSC‐sEVs at single particle‐resolution. Three sEVs traditional surface markers (CD9, CD63, and CD81), one validated protein (PODXL), and three specific surface antigens on PSC (SSEA4, Tra‐1‐60, and Tra‐1‐81) were selected for surface marker detection. (a‐d) NanoFCM analysis for the sEVs traditional surface markers including CD9, CD63, CD81, and combination of CD9/CD63/CD81 on PSC‐sEVs. Left panel, histograms showing the fluorescence intensity. Right panel, bar graphs showing the positive rates of surface markers. (e‐h) NanoFCM analysis for the indicated surface markers including PODXL, SSEA4, Tra‐1‐60, and Tra‐1‐81 on PSC‐sEVs. Left panel, histograms showing the fluorescence intensity. Right panel, bar graphs showing the positive rates of surface markers. PSC‐sEVs, pluripotent stem cell‐derived sEVs.

Article Snippet: The following antibodies were used for immunofluorescent staining: Alexa Fluor 488 (AF488) Mouse Anti‐Human CD9 (NanoFCM, NHA009‐A488‐50T, 1:10), AF488 Mouse Anti‐Human CD63 (NanoFCM, NHA063‐A488‐50T, 1:10), FITC Mouse Anti‐Human CD81 (NanoFCM, NHA‐FITC‐50T, 1:10), AF488 anti‐human PODXL Antibody (Abcam, ab208254, 1:20), AF488 anti‐human SSEA‐4 Antibody (BioLegend, 330411, 1:20), AF488 anti‐human TRA‐1‐81 Antibody (BioLegend, 330709, 1:20), AF488 anti‐human TRA‐1‐60‐R Antibody (BioLegend, 330613, 1:20).

Techniques: Single Particle, Marker, Fluorescence, Derivative Assay

Journal: Journal of Extracellular Vesicles

Article Title: Identification of specific markers for human pluripotent stem cell‐derived small extracellular vesicles

doi: 10.1002/jev2.12409

Figure Lengend Snippet: Validation of specificity of the markers with Immunofluorescent staining at single particle‐resolution. Seven non‐PSC cell lines derived sEVs were used for validation of specificity of the putative surface markers for PSC‐sEVs. (a‐d) Marker phenotyping analysis with NanoFCM for the sEVs traditional surface markers including CD9, CD63, CD81 and combination of CD9/CD63/CD81 on sEVs. Left panel, histograms showing the fluorescence intensity. Right panel, bar graphs showing the positive rates of surface markers. (e‐f) Marker phenotyping analysis with NanoFCM for the PODXL and SSEA4 on sEVs, showing the positive rates of PODXL and SSEA4 in non‐PSC‐sEVs were much lower than those in PSC‐sEVs. Left panel, histograms showing the fluorescence intensity. Right panel, bar graphs showing the positive rates of surface markers. PSC‐sEVs, pluripotent stem cell‐derived sEVs.

Article Snippet: The following antibodies were used for immunofluorescent staining: Alexa Fluor 488 (AF488) Mouse Anti‐Human CD9 (NanoFCM, NHA009‐A488‐50T, 1:10), AF488 Mouse Anti‐Human CD63 (NanoFCM, NHA063‐A488‐50T, 1:10), FITC Mouse Anti‐Human CD81 (NanoFCM, NHA‐FITC‐50T, 1:10), AF488 anti‐human PODXL Antibody (Abcam, ab208254, 1:20), AF488 anti‐human SSEA‐4 Antibody (BioLegend, 330411, 1:20), AF488 anti‐human TRA‐1‐81 Antibody (BioLegend, 330709, 1:20), AF488 anti‐human TRA‐1‐60‐R Antibody (BioLegend, 330613, 1:20).

Techniques: Biomarker Discovery, Staining, Single Particle, Derivative Assay, Marker, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Identification of specific markers for human pluripotent stem cell‐derived small extracellular vesicles

doi: 10.1002/jev2.12409

Figure Lengend Snippet: Validation of sEVs preparations via DGUC. UC samples of PSC‐sEVs were further purified with DGUC and then analyzed with Western Blot and NanoFCM. (a) Workflow of sucrose density gradient ultracentrifugation. (b) Western Blot analysis of TSG101 in PSC‐sEVs from the distinct DGUC fractions showing that sEVs were enriched in F6‐F8. (c‐d) Western Blot analysis of the putative markers, including PODXL, LIN28A, OCT4, and Dnmt3A, in PSC and PSC‐sEVs preparations via DGUC. **, p <0.01; ***, p <0.001; #, p <0.0001. ns, no significant difference. (e‐j) Marker phenotyping analysis with NanoFCM for the sEVs surface markers including CD9, CD63, CD81, combination of CD9/CD63/CD81, PODXL, and SSEA4 on sEVs. Left panel, histograms showing the fluorescence intensity. Right panel, bar graphs showing the positive rates of surface markers. DGUC, density gradient ultracentrifugation; UC, ultracentrifugation; PSC‐sEVs, pluripotent stem cell‐derived sEVs.

Article Snippet: The following antibodies were used for immunofluorescent staining: Alexa Fluor 488 (AF488) Mouse Anti‐Human CD9 (NanoFCM, NHA009‐A488‐50T, 1:10), AF488 Mouse Anti‐Human CD63 (NanoFCM, NHA063‐A488‐50T, 1:10), FITC Mouse Anti‐Human CD81 (NanoFCM, NHA‐FITC‐50T, 1:10), AF488 anti‐human PODXL Antibody (Abcam, ab208254, 1:20), AF488 anti‐human SSEA‐4 Antibody (BioLegend, 330411, 1:20), AF488 anti‐human TRA‐1‐81 Antibody (BioLegend, 330709, 1:20), AF488 anti‐human TRA‐1‐60‐R Antibody (BioLegend, 330613, 1:20).

Techniques: Biomarker Discovery, Purification, Western Blot, Marker, Fluorescence, Derivative Assay

Journal: Scientific Reports

Article Title: Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition

doi: 10.1038/srep04701

Figure Lengend Snippet: (a), Experimental schematic for immunostaining the endometrial epithelium with an anti-CD9 mAb. After estrus-stage mice were identified by a vaginal smear test, the uterus was isolated and examined by immunohistochemical analysis. (b), As depicted from the left, the endometrium includes epithelial layers (EL), a basement membrane (BM), and stromal layers (SL). The endometrial epithelium was incubated with the anti-CD9 mAb and then an Alexa Fluor 488-labeled secondary antibody. UC, uterine cavity. Arrowheads, CD9-reduced apical regions. Scale bar, 20 μm. (c), Experimental schematic for immunoblotting of uterine secretions at each stage of the estrous cycle, and immuno-electron microscopic analysis of uterine secretions at the estrus stage. (d), Immunoblotting of uterine secretions collected from each stage of the estrous cycle. (e), Immuno-electron microscopic images of uterine secretions at the estrus stage. (f) and (g), Enlarged images of boxes in (e). Scale bars, 100 nm. (h), Schematic of the two types of CD9.

Article Snippet: For immunohistochemistry and immunoblotting, a rat anti-mouse CD9 monoclonal antibody (mAb) (clone KMC8) and mouse anti-human CD9 mAb (clone ALB6) were purchased from BD Biosciences (San Jose, CA), and a mouse anti-mouse vascular endothelial growth factor (VEGF) mAb (clone RM0009-2G02) was purchased from Abcam.

Techniques: Immunostaining, Isolation, Immunohistochemical staining, Incubation, Labeling, Western Blot

Journal: Scientific Reports

Article Title: Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition

doi: 10.1038/srep04701

Figure Lengend Snippet: (a), Uterine secretions were collected from the uterine cavity and examined by immunoblotting. Human samples were immunoblotted with anti-CD9 and anti-β-actin-mAbs, and stained with Coomassie brilliant blue for detection of albumin. The number of samples is shown. (b), The presence of CD9 in uterine flushing in recurrent implantation failure (RIF) patients (n = 115) and the control patients (n = 56). A significant difference among the mean values with asterisk was observed (P < 0.05). (c), The association of the CD9 presence with a history of dilation and curettage (D&C) in the RIF patients (D&C[+], n = 22; D&C[−], n = 49). A significant difference among the mean values with asterisk was observed (P < 0.05). (d), The association of the CD9 presence with endometrial thickness in the RIF patients. When endometrium width measured by vaginal ultrasound was less than 8.5 mm at the mid luteal phase, the endometrium was categorized as thin endometrium (n = 22). When the width was 8.5 mm or more than that, the endometrium was categorized normal-width endometrium (n = 69). A significant difference among the mean values with asterisk was observed (P < 0.05). (e), The association of the CD9 presence with prognosis (miscarriage rates) in the RIF patients. The RIF patients were classified into four groups: those with CD9 (+) and normal endometrium (n = 7), those with CD9 (+) and thin endometrium (n = 6), those with CD9 (−) and normal endometrium (n = 20), and those with CD9 (−) and thin endometrium (n = 9). A significant difference among the mean values that have different superscripts was observed (P < 0.05). The endometrial thickness was measured by ultrasound and magnetic resonance imaging at the site indicated with the line in (a), and double-headed arrows in (f). (f), Schematic explanation of a hypothetical relationship between the absence of CD9 and endometrial thinning in the RIF patients.

Article Snippet: For immunohistochemistry and immunoblotting, a rat anti-mouse CD9 monoclonal antibody (mAb) (clone KMC8) and mouse anti-human CD9 mAb (clone ALB6) were purchased from BD Biosciences (San Jose, CA), and a mouse anti-mouse vascular endothelial growth factor (VEGF) mAb (clone RM0009-2G02) was purchased from Abcam.

Techniques: Western Blot, Staining, Magnetic Resonance Imaging

Journal: Scientific Reports

Article Title: Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition

doi: 10.1038/srep04701

Figure Lengend Snippet: (a), Experimental schematic for examining the litter size of Cd9 −/− TG female mice. (b), Age-independent reduction of the litter size of Cd9 −/− TG mice. (c), Reduction of the litter size dependent on parturition in Cd9 −/− TG mice. Parenthesis indicates the number of examined mice. Values are the mean ± SEM. (d) and (e), Histochemical analysis of endometrial repair in Cd9 −/− TG and Cd9 +/+ mice after parturition (the day of parturition = day 0). (f), Experimental schematic for in vitro wound healing assays of the endometrial epithelium of Cd9 −/− TG mice. After the uterus was isolated from Cd9 −/− TG mice at the estrus stage, collagenase was injected to the intrauterine cavity to collect the epithelial cells. (g), Wounded epithelial cells after scratching the monolayer with a pipette tip. Scale bars, 150 μm. (h), Graph of the wound width of wounded epithelial cells. Values are the mean ± SEM.

Article Snippet: For immunohistochemistry and immunoblotting, a rat anti-mouse CD9 monoclonal antibody (mAb) (clone KMC8) and mouse anti-human CD9 mAb (clone ALB6) were purchased from BD Biosciences (San Jose, CA), and a mouse anti-mouse vascular endothelial growth factor (VEGF) mAb (clone RM0009-2G02) was purchased from Abcam.

Techniques: In Vitro, Isolation, Injection, Transferring

Journal: Scientific Reports

Article Title: Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition

doi: 10.1038/srep04701

Figure Lengend Snippet: (a), Comparison of the amount of VEGF in uterine secretions of Cd9 −/− TG mice at the estrus stage using a multiplex suspension array. (b), Immunoblotting of uterine secretions at the estrus stage in Cd9 −/− TG and Cd9 +/+ mice. (c), Immunohistochemical observation of the endometrial epithelium in Cd9 −/− TG and Cd9 +/+ mice. White arrowheads indicate the uterine cavity. Scale bars, 20 μm. (d), Electron microscopic images of endometrial epithelial cells in Cd9 −/− TG and Cd9 +/+ mice. Left panels, endometrial epithelial cells of Cd9 −/− TG and Cd9 +/+ mice at the estrus stage. Middle panels, enlarged images of the boxes in the left panels. Right panels, enlarged images of the boxes in the middle images. Hollow arrowheads indicate the secreted materials in Cd9 −/− TG mice. Arrowheads indicate the outer membrane consisting of lipid bilayers in Cd9 +/+ mice. Scale bars, 500 nm. (e), Length of microvilli. Left graph, endometrial epithelial cells at the estrus stage. Right graph, endometrial epithelial cells at the metestrus stage. Values are the mean ± SEM.

Article Snippet: For immunohistochemistry and immunoblotting, a rat anti-mouse CD9 monoclonal antibody (mAb) (clone KMC8) and mouse anti-human CD9 mAb (clone ALB6) were purchased from BD Biosciences (San Jose, CA), and a mouse anti-mouse vascular endothelial growth factor (VEGF) mAb (clone RM0009-2G02) was purchased from Abcam.

Techniques: Multiplex Assay, Western Blot, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Absence of CD9 reduces endometrial VEGF secretion and impairs uterine repair after parturition

doi: 10.1038/srep04701

Figure Lengend Snippet: (a), Experimental schematic for treatment of the endometrial epithelium of Cd9 −/− TG mice with VEGF. (b), Ventral view of the horns of a uterus isolated from a Cd9 −/− TG mouse at 1 week after VEGF-linked microparticles were injected into the uterine cavity. Scale bar, 5 mm. (c), Endometrial epithelium treated with VEGF or BSA as a control. Lower panels, enlarged images of boxes in the upper panels. Double-headed arrows indicate the endometrial thickness. Hollow arrowheads indicate the re-epithelialized layers. The dotted circle indicates the fused site of endometrial stromal layers without epithelium with a mixture of microparticles and immune cells. Scale bars, 300 μm in upper panels and 100 μm in lower panels. (d), The rate of re-epithelialized sites in the Cd9 −/− TG endometrium treated with VEGF or BSA. (e), Thickness of the endometrium treated with VEGF or BSA. Values are the mean ± SE. (f), Schematic model of re-epithelialization of the endometrium treated with VEGF. The endometrial thickness was measured at the site indicated with double-headed arrows. SL, stromal layers; Myo, myometrium. (g), Schematic model of CD9 and VEGF secretion from epithelial cells. CD9-mediated VEGF release contributes to endometrial re-epithelialization.

Article Snippet: For immunohistochemistry and immunoblotting, a rat anti-mouse CD9 monoclonal antibody (mAb) (clone KMC8) and mouse anti-human CD9 mAb (clone ALB6) were purchased from BD Biosciences (San Jose, CA), and a mouse anti-mouse vascular endothelial growth factor (VEGF) mAb (clone RM0009-2G02) was purchased from Abcam.

Techniques: Isolation, Injection

Journal: American Journal of Translational Research

Article Title: The relationship between amniotic fluid miRNAs and congenital obstructive nephropathy

doi:

Figure Lengend Snippet: Representative FACS analysis plots for CD9 expression on exosome-bead conjugates.

Article Snippet: Chemicals and antibodies FITC Mouse Anti-Human CD24 (555427, HAS, 1:50, BD), PE Mouse Anti-Human CD9 (555372, HAS, 1:50, BD), CD24 antibody (sc-11406, SANTA CRUZ, 1:200), CD9 antibody (sc-9148, SANTA, 1:200).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: The relationship between amniotic fluid miRNAs and congenital obstructive nephropathy

doi:

Figure Lengend Snippet: Representative western blot images of CD24 and CD9 expression in purified exosomes.

Article Snippet: Chemicals and antibodies FITC Mouse Anti-Human CD24 (555427, HAS, 1:50, BD), PE Mouse Anti-Human CD9 (555372, HAS, 1:50, BD), CD24 antibody (sc-11406, SANTA CRUZ, 1:200), CD9 antibody (sc-9148, SANTA, 1:200).

Techniques: Western Blot, Expressing, Purification

Journal: bioRxiv

Article Title: Targeted delivery of mRNA to the heart via extracellular vesicles or lipid nanoparticles

doi: 10.1101/2025.01.25.634881

Figure Lengend Snippet: 3 µg of LNP- VEGF-A mRNA was administered to cardiac progenitor cells (CPCs), human umbilical vein endothelial cells (HUVECs) and a human lung epithelial cell line (HTBs). ( A ) A schematic illustration showing the uptake of LNP-mRNA by the recipient cells, escape from the endosomes for translation into protein. (B) Quantification of VEGF-A mRNA by qPCR, in the lysates of recipient cells ( n = 6). (C) Quantification of VEGF-A protein by ELISA, in supernatants of recipient cells ( n = 6). Statistically significant differences between LNP-treated and untreated samples were evaluated by applying Mann-Whitney U-test. Significant differences (**** p < 0.0001) were observed between LNP-treated and untreated HUVECs and CPCs. No significant differences were observed between LNP-treated and untreated HTBs. UNT: untreated cells. 24h post LNP-mRNA uptake by cells, the conditioned media was collected, and the extracellular vesicles (EVs) secreted from these cells were isolated and purified by size exclusion chromatography (SEC), as described in the methods. (D) illustration showing the cellular route of mRNA loading into EVs and secretion. (E) Scheme showing the isolation of EVs by qEV/10 collum for size exclusion chromatography. (F) 12 fractions of SEC-EVs were collected and the presence of VEGF-A mRNA and protein in fractions of HTB-EVs, was determined by qPCR and ELISA, respectively. SEC-EVs carried substantial amount of exogenous VEGF-A mRNA, which was only possible to identify in fractions F1-6. (G) The total amounts of VEGF-A mRNA in SEC-EVs secreted from different cell types: HTBs, HUVECs, and CPCs. Statistically significant differences between LNP-treated and untreated samples were evaluated by applying Mann-Whitney U-test. Significant differences (**** p < 0.0001). (H) Experimental scheme showing the immunocapture of SEC-EVs. SEC-EVs were first captured by anti-C63 antibody and then these CD63 positive EVs were further captured by CD9, conjugated with PE. (I) identification of EV markers; CD63 and CD9 in SEC-EVs. A 69 % of CD63 + HTB-EVs were also positive for CD9. A 90 % of CD63 + HUVEC-EVs of cells were also positive for CD9. CPC-EVs were positive for CD63, however only 0.56 % of CD63 EVs were positive for CD9. Unstained samples were used as negative control where no signals were detected. (J) . Transmission electron microscopy of HTB-EVs, HUVEC-EVs, and CPC-EVs. Scalebar: 100nm.

Article Snippet: After the final wash, the immobilized CD63 + EVs were suspended in 120 μL of BSA-PBS isolation buffer and then further stained with a mouse anti-human PE-CD9 antibody (BD PharmingenTM, cat.#: 555372).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Isolation, Purification, Size-exclusion Chromatography, Negative Control, Transmission Assay, Electron Microscopy