Journal: Viruses

Article Title: Cargo and Biological Properties of Extracellular Vesicles Released from Human Adenovirus Type 4-Infected Lung Epithelial Cells

doi: 10.3390/v17101300

Figure Lengend Snippet: Characterization of the EV populations isolated from the mock- and AdV-infected A549 cells. ( A ) Phase contrast images of the A549 cells showing the magnitude of cytopathic effect at 24 h post-infection. Cells were infected at MOI = 10. Scale bars = 100 µm. ( B ) The representative Western blot analysis showing enrichment of the EV markers CD63, CD9, CD81, Alix in EVs-Ctr and EVs-AdV, compared to their cells of origin (cell lysates). The ER marker GRP94 was used to assess the presence of non-EV contaminants. Images are representative of three different experiments. ( C ) The TEM images of EVs isolated by magnetic immunocapture form the supernatants of mock-infected cells (Ctr-EVs) or HAdV-E4-infected (AdV-EVs) A549 cells. The magnetic beads are recognizable by their opaque appearance and uniform size of 50 nm (black arrows). Scale bars = 200 nm. ( D ) The TEM image of the purified HAdV-E4 particles (white arrows). Scale bar = 200 nm. ( E ) The fluorescent nanoparticle tracking analysis of Ctr-EVs (left panel), AdV-EVs (middle panel), and magnetic beads alone (right panel).

Article Snippet: EVs were isolated from the concentrated medium with EV isolation kits Pan (human), or CD81 human (Cat # 130-111-572 and 130-110-914, Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instructions.

Techniques: Isolation, Infection, Western Blot, Marker, Magnetic Beads, Purification

Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: Colocalization of endocytic BDNF with CD63 or Rab11. ( A ) Representative fluorescence images showing the colocalization of QD-BDNF with CD63- or Rab11-EGFP in astrocytes. QD-BDNF was added for 30–12 min to cultured astrocytes transfected with CD63-EGFP or Rab11-EGFP. Yellow arrowheads indicate QD-BDNF colocalized with the corresponding vesicular markers. Scale bar = 5 μm. ( B ) Bar graphs depict average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). Dotted line: average colocalization ratio between QD-BDNF and MitoTracker (Mito.) as a negative control. *P < 0.05. N = 12–15 cells (194–2,782 QD particles). ( C ) Representative fluorescence images showing the colocalization of QD-BDNF with endogenous CD63 or Rab11 in astrocytes. Yellow arrowheads indicate QD-BDNF colocalized with the corresponding vesicular markers stained with anti-CD63 or anti-Rab11. Scale bar = 5 μm. ( D ) Bar graphs depict average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). Dotted line: average colocalization ratio between QD-BDNF and MitoTracker (Mito.) as a negative control. *P < 0.05. N = 10–16 cells (784-2,110 QD particles).

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Fluorescence, Cell Culture, Transfection, Negative Control, Staining

Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: EV-mediated release of endocytosed BDNF from astrocytes. ( A ) Schematic diagram of the experiment for detecting BDNF-EGFP derived from Neuro2A cells in astrocytic EVs. ( B ) Dot blot showing EGFP expression in cell lysates from nontransfected (non. ; negative control) or BDNF-EGFP-transfected Neuro2a cells. ( C ) Dot blotting was used to detect protein expression in cell lysates and EVs extracted from the media of astrocytes treated with ATP or vehicle after being supplied with media from nontransfected or BDNF-EGFP-transfected Neuro2a cells. Antibodies targeting general markers of the endoplasmic reticulum (ER; an EV-negative marker, calregulin) or EVs (TSG101) were used. ( D ) Representative QD-BDNF kymographs from astrocytes were generated via ImageJ/FIJI and obtained after treatment with vehicle or GW4869 (20 µM) for 1 h prior to time-lapse imaging. Red bar: ATP (100 µM) treatment. Arrow heads: disappearance of QD-BDNF fluorescence. ( E ) Average percentages of ATP-induced QD-BDNF secretion events from vesicles in astrocytes pretreated with either vehicle or GW4869. ****P < 0.0001. N = 9–11 cells (vehicle = 201; GW4869 = 468 QD particles). ( F ) Schematic diagram showing overall process of immunocapture of CD63-positive EVs released from ACM using anti CD63-linked magnetic beads. ( G - H ) Bar graphs depict average concentration of BDNF from ACM using ELISA assay with anti-BDNF (G) or BDNF-EGFP with anti-GFP (H). N = 4 ACM batches derived from different astrocyte cultures. Each dot indicates the result measured from individual batch.

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Derivative Assay, Dot Blot, Expressing, Negative Control, Transfection, Marker, Generated, Imaging, Fluorescence, Magnetic Beads, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: Vamp3-dependent secretion of QD-BDNF from CD63-positive vesicles. ( A ) Representative QD-BDNF kymographs generated from CD63-EGFP-transfected astrocytes. Arrow heads: disappearance of CD63-EGFP-positive QD-BDNF fluorescence. ( B ) Average fractions of secreted QD-BDNF particles with (+) or without CD63 (-) among total secreted QD-BDNF particles. N = 24 cells (232 secreted QD particles). ( C ) Average percentages of ATP-induced QD-BDNF secretion events from vesicles with (+) or without CD63 (-). **P < 0.01, ****P < 0.0001. N = 10–11 cells ( siSCR : 11 cells, 508 QD particles; siVamp3 : 10 cells, 308 QD particles). siSCR : 3 independent experiments with 3–4 cells (107–224 QD particles per experiment); siVamp3 : 3 independent experiments with 3–4 cells (91–114 QD particles per experiment). ( D ) Representative fluorescence images showing the colocalization of QD-BDNF with CD63-EGFP in astrocytes treated with siSCR or siVamp3 . Yellow arrowheads indicate QD-BDNF colocalized with CD63-EGFP. Scale bar = 5 μm. ( E ) Average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). *P < 0.05. N = 12 cells (1,737–1,962 QD particles). ( F ) Representative fluorescence images showing the colocalization of QD-BDNF with endogenous CD63 in astrocytes treated with siSCR or siVamp3 . Yellow arrowheads indicate QD-BDNF colocalized with anti-CD63 signals. Scale bar = 5 μm. ( G ) Average colocalization ratios (number of colocalized QD-BDNF particles among total QD-BDNF particles). *P < 0.05. N = 20–29 cells (2,150-2,464 QD particles).

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Generated, Transfection, Fluorescence

Journal: Scientific Reports

Article Title: Recycling of endocytic BDNF through extracellular vesicles in astrocytes

doi: 10.1038/s41598-025-86200-x

Figure Lengend Snippet: Key resource table.

Article Snippet: EV isolation Kit CD63, mouse , Miltenyi Biotec , 130–117-041 , .

Techniques: Control, Virus, Protease Inhibitor, Plasmid Preparation, Software, Staining, Membrane, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 1 Graphic illustration of the different combinations of sample material and EV isolation protocols applied to the EV Neuro assay. For further details, see text. Abbreviations: HA: primary human astrocytes; GB: glioblastoma; HC: healthy control; MS: multiple sclerosis; MAD: mild cognitive impairment/ Alzheimer’s disease/depression; SEC: size-exclusion chromatography; UC: ultracentrifugation

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Isolation, Control, Size-exclusion Chromatography

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 2 Characterization of EVs derived from glioblastoma cells and astrocytes. a Size distribution of EVs separated from cell culture supernatant of LN18, LN229, and NCH82 glioblastoma cell lines as well as from primary human astrocytes (HA). b Western blot analysis of CD9, CD63, CD81, Syntenin (EV markers) and Calnexin (non-EV marker) in EVs separated from cell culture supernatant of glioblastoma cells and primary astrocytes. c EV Neuro median signal intensities for LN18, LN229, NCH82, and HA EVs. d Relative signal intensities of values for cell derived EVs shown in (c). Relative signals are calculated by dividing the signal intensity of a target by the sum of intensities revealed from all markers (total intensity) and presented in percentages. Bars reflect representative marker profiles (n = 1–3 biological replicates). Arrow heads indicate markers that appear elevated in cell lines compared to primary astrocytes

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Derivative Assay, Cell Culture, Western Blot, Marker

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 3 Titration of EVs derived from glioblastoma cells. EV Neuro median signal intensities for increasing total numbers of NCH82 EVs (a) and LN18 EVs (b) as determined by NTA particle count. The number of 2.8 × 107 particles (turquoise) represents the input of 120 µl of pre-cleared cell culture supernatant (10,000 × g) without prior EV enrichment. Other particle inputs (blue and red) were achieved by EV enrichment via dUC. Bars reflect marker profiles of one experiment each

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Titration, Derivative Assay, Cell Culture, Marker

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 4 Profiling of serum EVs from GB patients and healthy controls. a Relative EV Neuro signal intensities for EVs separated from the serum of GB patients or HC by SEC followed by UC calculated as signal of target divided by the total signal of all markers (in %). Bars represent mean values and error bars indicate the 95% confidence interval. Asterisks mark statistically significant differences between GB and HC. Only significant alterations of targets that were consistently detected above background in all individuals are marked. *=p < .05, **=p < .01, ***=p < .001. (b) tSNE on data from (a) stratified by condition (color). c Heatmap visualization of selected targets from (a) including hierarchical clustering for targets as well as subjects

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques:

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 6 Comparison of plasma EVs from MS patients in a relapse versus in a stable disease phase. Relative EV Neuro signal intensities as signal of target divided by the total signal of all markers (in %) for immuno-affinity captured CD63+EVs (a) and CD81+EVs (b). Bars represent mean values and error bars indicate the 95% confidence interval. Asterisks mark statistically significant differences between stable phase and disease relapse. Only significant alterations of targets that were consistently detected above background in all individuals are marked. *=p < .05, **=p < .01

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Comparison, Clinical Proteomics

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 7 Phenotyping of plasma EVs from MAD patients and comparison of all plasma samples. a Relative EV Neuro signal intensities for EVs separated from the plasma of MAD patients by immuno-affinity capture (anti-CD81) and magnetic separation. Bars represent mean values and error bars indicate the 95% confidence intervals (n = 8 patients). b tSNE of relative signal intensities from all plasma-derived EV samples analyzed (MS, MAD, HC) stratified by condition (color) and isolation (shape). c Heatmap visualization of relative signals for selected targets from all plasma samples analyzed including hierarchical clustering for targets as well as subjects (BCL = lab of blood collection)

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Clinical Proteomics, Comparison, Derivative Assay, Isolation