Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Enhanced Workflow for Urinary Extracellular Vesicle Isolation Using Membrane-Sensing Peptides

doi: 10.64898/2026.05.21.726982

Figure Lengend Snippet: Comparative analysis of tetraspanin expression and co-localization in isolated uEVss. A) MACSPlex analysis in pre-cleared urine samples (yellow bars) and in MSP-isolated uEVss (purple bars). Data were normalized to the median fluorescence intensity of all detectable markers; B) CD9 Median Fluorescence Intensity in the small EV isolated measured using HS-FCM technology comparing dUC and MSP; C) Schematic representation of the single-tetraspanin SiMoA assay; corresponding plots show the average enzyme per bead (AEB) signal for individual tetraspanins (CD9, CD63, and CD81) in uEVss isolated by dUC, SEC, and MSP-functionalized beads (N = 5). B) Sketch of EV-labeling with mAbs anti-tetraspanin for F-NTA analysis; percentage of events that colocalized for two different tetraspanin simultaneously.

Article Snippet: For each sample, 3 x 10 8 particles (calculated by NTA quantification) were incubated for 15 minutes protected from light with 0,75 μg/ml of Phycoerythrin (PE)-conjugated anti-CD9 antibody (Cat. No. 130-124-758, Miltenyi Biotec), reaching a total volume of 100 μl with filtered (0,1 μm pores) PBS.

Techniques: Expressing, Isolation, Fluorescence, Labeling

Journal: Alzheimer's Research & Therapy

Article Title: Stress-induced alteration of small extracellular vesicles drives amyloid-beta sequestration and exacerbates Alzheimer’s disease pathogenesis

doi: 10.1186/s13195-026-02028-1

Figure Lengend Snippet: Graphical representation of workflow for isolation and characterization of Plasma-derived EVs ( A ). Characterization of isolated EVs through transmission electron microscopy ( B ), Scale bar= 100 nm; and confocal microscopy ( C ), Scale bar= 20 μm. Nanoparticle Analysis (NTA) showing the size distribution of EVs (nm) and particle concentration (particle/ml) ( D ); Western blot of EVs markers (CD9, CD63, CD81, Tsg101) and protein co-isolate (Apolipoprotein-B) where Plasma (P) and F [ – ] = SEC Fractions collected ( E )

Article Snippet: sEVs and Aβ42 suspension was labeled using Human CD9 Alexa Fluor ® 488-conjugated Antibody (R&D systems, FAB1880G) for sEVs and Alexa Fluor ® 647 Anti-beta Amyloid 1–42 antibody [mOC64] (Abcam, ab300742) in final concentration of 2%(v/v) and 0.5%(v/v) of the total suspension respectively. sEVs and the aggregate suspension were incubated for 2 h with both antibodies at 25 °C before mounting on labolene-cleaned glass slides (Blue Star) and covered with 18 mm;10Gms glass covers (Blue Star), and were imaged on Zeiss LSM980 system using the 40X objective.

Techniques: Isolation, Clinical Proteomics, Derivative Assay, Transmission Assay, Electron Microscopy, Confocal Microscopy, Concentration Assay, Western Blot

Journal: Alzheimer's Research & Therapy

Article Title: Stress-induced alteration of small extracellular vesicles drives amyloid-beta sequestration and exacerbates Alzheimer’s disease pathogenesis

doi: 10.1186/s13195-026-02028-1

Figure Lengend Snippet: Graphical representation of workflow A . In-vitro assessment of amyloid-β (Unaggregated) and EV association. Imaging was performed at three key stages: Control (Without any stresses), Sonication only, and Sonication and agitation combined. TEM micrograph shows morphological characterization of different Aβ aggregated structures formed in the presence of EVs ( B ), Scale bar= 100 nm; CFM images for respective groups ( C )and Colocalization analysis ( D ) showed maximum PCC in the post-sonication group (PCC = 0.73). Colour yellow is the merged signal of EVs (Green) and Aβ (red). Scale bar= 20 μm. PCC between CD9-Alexa 488 EVs and AβSA-Alexa 647. Control: 0.033 ± 0.005; Sonicated: 0.780 ± 0.046; Sonicated & Agitated: 0.499 ± 0.066 ( n = 3). One-way ANOVA: p < 0.0001 (E)

Article Snippet: sEVs and Aβ42 suspension was labeled using Human CD9 Alexa Fluor ® 488-conjugated Antibody (R&D systems, FAB1880G) for sEVs and Alexa Fluor ® 647 Anti-beta Amyloid 1–42 antibody [mOC64] (Abcam, ab300742) in final concentration of 2%(v/v) and 0.5%(v/v) of the total suspension respectively. sEVs and the aggregate suspension were incubated for 2 h with both antibodies at 25 °C before mounting on labolene-cleaned glass slides (Blue Star) and covered with 18 mm;10Gms glass covers (Blue Star), and were imaged on Zeiss LSM980 system using the 40X objective.

Techniques: In Vitro, Imaging, Control, Sonication

Journal: Alzheimer's Research & Therapy

Article Title: Stress-induced alteration of small extracellular vesicles drives amyloid-beta sequestration and exacerbates Alzheimer’s disease pathogenesis

doi: 10.1186/s13195-026-02028-1

Figure Lengend Snippet: Comparison of interaction between EV and small amyloid-β aggregates ( A ) and big amyloid-β aggregates/Fibrils ( B ) at low temperature 4℃. The TEM, Scale bar= 100 nm;, and CFM micrograph shows the EVs sequestering the SA group as opposed to no association between BA/Fibril B . Graphical representation of workflow C . CFM image showing Alexa-Fluor-488 CD9 (Green) and Alexa-Fluor-647Amyloid-β (Red) signals of: EVs only; Aβ; and EVs and Aβ together D . Scale bar= 20 μm. Fluorescence intensity per cell was measured as Mean Fluorescence intensity/Area of ROI. Each condition included 3 cells per dish with a total of n = 9 cells per group. Statistical analysis using one-way ANOVA (Kruskal–Wallis test) indicated a significant difference ( p < 0.001) E . Cell viability ( F ) 10,000cells/well were seeded and included 4 wells per plate with a total of n = 4. Statistical analysis using one-way ANOVA and multiple comparisons showing a highly significant effect ( p < 0.00001)

Article Snippet: sEVs and Aβ42 suspension was labeled using Human CD9 Alexa Fluor ® 488-conjugated Antibody (R&D systems, FAB1880G) for sEVs and Alexa Fluor ® 647 Anti-beta Amyloid 1–42 antibody [mOC64] (Abcam, ab300742) in final concentration of 2%(v/v) and 0.5%(v/v) of the total suspension respectively. sEVs and the aggregate suspension were incubated for 2 h with both antibodies at 25 °C before mounting on labolene-cleaned glass slides (Blue Star) and covered with 18 mm;10Gms glass covers (Blue Star), and were imaged on Zeiss LSM980 system using the 40X objective.

Techniques: Comparison, Fluorescence

Journal: Alzheimer's Research & Therapy

Article Title: Stress-induced alteration of small extracellular vesicles drives amyloid-beta sequestration and exacerbates Alzheimer’s disease pathogenesis

doi: 10.1186/s13195-026-02028-1

Figure Lengend Snippet: Graphical representation of workflow A . Immunohistological staining images of APP mouse brain sections with CD9 (EVs) and ThT (plaques) showing typical Amyloid- plaques with discrete CD9 signals ( B ) and respective colocalization coefficient D . In-vitro dual staining experimental images: ( C ) APP mice brain sections immunohistological staining with CD9 (antibody) and ThT stained Aβ. ( n = 3 mice brains) (Scale bar 20 μm; Zeiss Confocal microscope and NIS-Elements BR 4.30.00.64-bit fluorescence microscope). Images were captured at (For ThT; λex = 450 nm and λem = 490 nm. CD9-Rodamine TRITC-conjugated; λex = 550 nm and λem = 570 nm). Scale bar= 20 μm

Article Snippet: sEVs and Aβ42 suspension was labeled using Human CD9 Alexa Fluor ® 488-conjugated Antibody (R&D systems, FAB1880G) for sEVs and Alexa Fluor ® 647 Anti-beta Amyloid 1–42 antibody [mOC64] (Abcam, ab300742) in final concentration of 2%(v/v) and 0.5%(v/v) of the total suspension respectively. sEVs and the aggregate suspension were incubated for 2 h with both antibodies at 25 °C before mounting on labolene-cleaned glass slides (Blue Star) and covered with 18 mm;10Gms glass covers (Blue Star), and were imaged on Zeiss LSM980 system using the 40X objective.

Techniques: Staining, In Vitro, Microscopy, Fluorescence

Journal: Alzheimer's Research & Therapy

Article Title: Stress-induced alteration of small extracellular vesicles drives amyloid-beta sequestration and exacerbates Alzheimer’s disease pathogenesis

doi: 10.1186/s13195-026-02028-1

Figure Lengend Snippet: Graphical representation of workflow A . Circulating sEVs derived from Control, MCI and AD patient does not show colocalization signal B . Co-incubation with Aβ SA was performed only for control and AD EVs. Controls sEVs incubated with small amyloid-β aggregates C . AD sEVs, when incubated with small amyloid-β aggregates, colocalize (White arrows) at low temperature 4℃ D . Colour Yellow is the colocalised signal for sEVs (Green)and Aβ (Red). Scale bar= 20 μm. PCC ( E ) between CD9–Alexa Fluor 488 EVs and AβSA-Alexa Fluor 647, calculated using Costes thresholding (FIJI/JACoP). Control EVs: 0.033 ± 0.005; AD EVs: 0.780 ± 0.046 ( n = 3). One-way ANOVA: p < 0.0001. Percentage of AβSA associated with EVs ( F ), Control EVs: 2.87 ± 0.90%; AD EVs: 40.37 ± 1.98% (( n = 3; incubation maintained at 4 °C)). Unpaired t-test: p < 0.0001. AD ( n = 9), MCI ( n = 3), and non-demented controls ( n = 10)

Article Snippet: sEVs and Aβ42 suspension was labeled using Human CD9 Alexa Fluor ® 488-conjugated Antibody (R&D systems, FAB1880G) for sEVs and Alexa Fluor ® 647 Anti-beta Amyloid 1–42 antibody [mOC64] (Abcam, ab300742) in final concentration of 2%(v/v) and 0.5%(v/v) of the total suspension respectively. sEVs and the aggregate suspension were incubated for 2 h with both antibodies at 25 °C before mounting on labolene-cleaned glass slides (Blue Star) and covered with 18 mm;10Gms glass covers (Blue Star), and were imaged on Zeiss LSM980 system using the 40X objective.

Techniques: Derivative Assay, Control, Incubation