Journal: Molecular & Cellular Proteomics : MCP
Article Title: The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions
doi: 10.1016/j.mcpro.2023.100514
Figure Lengend Snippet: Characterization of extracellular vesicle (EVs) subsets, namely, small (S-EVs) or large (L-EVs), isolated from pig seminal plasma (SP) samples (n = 3; three ejaculates per sample; one ejaculate per male pig). A , violin plot displaying the total protein concentration in both SP-EV subsets. The dashed line shows the median and dotted lines the 25 to 75% quartiles. B , representative histogram of particle size distribution of S-EVs and L-EVs assessed by nanoparticle tracking analysis. C , particle size distribution of S-EVs and L-EVs analyzed by dynamic light scattering ( red , S-EVs; blue , L-EVs) in terms of intensity and volume. The black and gray lines represent the average of intensity size distribution of S-EVs and L-EVs, respectively. D , representative images of the morphology of S-EVs and L-EVs assessed by transmission electron microscopy. E , representative histogram of CFSE/CD63/HSP90β/ALB expression in S-EVs and L-EVs assessed by flow cytometry. ALB, albumin; CFSE, carboxyfluorescein succinimidyl ester; CNT, control; HSP90β, heat shock protein 90β.
Article Snippet: The EVs were cytometrically characterized following the International Society of Extracellular Vesicles recommendations (MIFlowCyt-EV, ( )) to identify their enrichment in proteins belonging to the three categories established by MISEV 2018 guidelines ( ): CD63 (Anti-CD63-FITC, Clone REA1055, Miltenyi Biotec) as “Category 1” protein (Transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes); HSP90β (anti-HSP90β-PE, ADI-SPA-844PE-050, Enzo Life Sciences) as “Category 2” protein (Cytosolic proteins recovered in EVs), and albumin (Anti-swine Albumin-FITC, CLFAG16140, Cedarlane) as “Category 3” protein (Major components of non-EVs coisolated structures).
Techniques: Isolation, Clinical Proteomics, Protein Concentration, Transmission Assay, Electron Microscopy, Expressing, Flow Cytometry, Control
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