Journal: STAR Protocols

Article Title: Protocol for the isolation and characterization of extracellular vesicles and particles from human and murine cell lines

doi: 10.1016/j.xpro.2026.104505

Figure Lengend Snippet: Cell culture and EVP isolation (A) MHCC97L cells are cultured in standard full medium, cell confluency is checked and maintained at ∼80%. (B) Remove the medium, cells are washed twice with 15 mL sterile 37°C PBS to remove residual serum and medium components. (C) MHCC97L cells are cultured in EVP-depleted full medium for 72 h, with cell confluency and morphology monitored every 24 h; for optimal EVP isolation, cultures are maintained at >95% confluency. (D) Schematic illustrating the isolation of small extracellular vesicles (sEV) and exomeres (EM) from cell-conditioned medium by sequential ultracentrifugation.

Article Snippet: Small extracellular vesicles (sEV; exosome) are lipid membrane-enclosed nanoparticles that are important for intercellular communication by transferring various biological cargoes.

Techniques: Cell Culture, Isolation, Sterility

Journal: STAR Protocols

Article Title: Protocol for the isolation and characterization of extracellular vesicles and particles from human and murine cell lines

doi: 10.1016/j.xpro.2026.104505

Figure Lengend Snippet: Characterization of small extracellular vesicles (sEV) and exomeres (EM) (A) Protein yield (μg per 10 7 cells) of fresh sEV and stored sEV; data are presented as mean ± SEM, P < 0.05 (Student’s t test). (B) Total protein yield (μg per 10 7 cells) of fresh EM and stored EM; data are presented as mean ± SEM, NS (not significant, Student’s t test). (C) Western blot analysis of total cell lysate (TCL), sEV, and EM fractions: exosome positive markers (Alix, TSG101, CD9), negative marker (GM130), and EM-enriched proteins (MAN2B1, GALNS); GAPDH serves as a loading control. (D) Representative transmission electron microscopy (TEM) images of isolated sEV (left) and EM (right); scale bar = 100 nm. (E) Nanoparticle tracking analysis (NTA) of sEV.

Article Snippet: Small extracellular vesicles (sEV; exosome) are lipid membrane-enclosed nanoparticles that are important for intercellular communication by transferring various biological cargoes.

Techniques: Western Blot, Marker, Control, Transmission Assay, Electron Microscopy, Isolation

Journal: Nature Communications

Article Title: Autophagic extracellular vesicles (AEVs) are distinct from exosomes and play crucial roles in viral infections

doi: 10.1038/s41467-025-67860-9

Figure Lengend Snippet: A Schematic presentation of the differential centrifugation protocol used to isolate autophagic extracellular vesicles (AEVs) from the cell culture supernatant. The majuscule A and B represented two methods for AEV isolation. Created in BioRender. Ruan, H. (2025) https://BioRender.com/pouij2q . B The percentage of AEVs with a size below 100 nm from supernatant using method A or method B were determined by NTA. 293 T cells were cultured under serum-starved condition for 24 h and the supernatant was collected. Data were presented as the mean ± SD of four independent experiments. p -values were determined by unpaired two tailed Student’s t tests. *** p = <0.0001. C The AEV fraction from serum-starved 293 T cells using method A or method B were collected and subjected to blotting for the indicated proteins. D The particle size distribution for p62-labelled AEVs were determined by NTA. The AEVs were isolated from serum-starved 293 T cells and incubated with p62 antibody conjugating Alexa Fluor 488. Data presented as mean ± SD from three independent experiments. GO enrichment analysis of proteins enriched in AEV fraction from serum-starved wild-type 293 T cells (starv-WT) relative to AEV fraction from normally cultured wild-type 293 T cells (nm-WT) with the top 20 terms for cellular component ( E ) and biological processes ( F ). Each group contained three independent biological replicates. G , H GO enrichment analysis of proteins decreased in AEV fraction from serum-starved Rab11 -KO 293 T cells (starv-KO) relative to AEV fraction from serum-starved WT 293 T cells (starv-WT) with the top 20 terms for cellular component ( G ) and biological processes ( H ). I Table of significantly changed proteins in different originated AEVs from quantitative proteomic analysis between starv-WT relative to nm-WT and starv-KO relative to strav-WT. Numbers in the table represented the multiples of change, indicating either an increase or a decrease. Source data are provided as a Source Data file.

Article Snippet: Previous studies revealed that interconnections between the autophagy process and small extracellular vesicle (sEV)/exosome production is closely connected .

Techniques: Centrifugation, Cell Culture, Isolation, Two Tailed Test, Incubation

Journal: Nature Communications

Article Title: Autophagic extracellular vesicles (AEVs) are distinct from exosomes and play crucial roles in viral infections

doi: 10.1038/s41467-025-67860-9

Figure Lengend Snippet: Upon autophagy induction, mature autophagosomes fuse with multivesicular bodies (MVBs) via Rab11 to form hybrid amphisome organelles. Autophagy cargos such as lipidated LC3B (LC3B-II) and p62, along with several ESCRT components, intermix with intraluminal vesicles through a process mediated by Rab13. This process involves intraluminal budding mechanisms that depend on the interaction between ESCRT-III components (CHMP4A and CHMP4B) and VPS4A/B. Subsequently, hybrid amphisomes release two subpopulations of small extracellular vesicles (sEVs) through Rab27a-dependent exocytosis: one consisting of autophagic extracellular vesicles (AEVs) containing autophagy cargos, ESCRT components and Rab13, the other being traditional exosomes. Created in BioRender. Ruan, H. (2025) https://BioRender.com/rhh7l0k .

Article Snippet: Previous studies revealed that interconnections between the autophagy process and small extracellular vesicle (sEV)/exosome production is closely connected .

Techniques: