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Extracellular vesicle isolation method from plasma samples. (A) Differential centrifugations followed by filtration and size exclusion chromatography (SEC). (B) Protein concentration (line) on the 14 SEC fractions, combined with western blot analysis using EV‐markers (ALIX and CD9) and <t>Apolipoprotein</t> A1 <t>(APO</t> A1) and B (APO B) as non‐EV markers on fractions 7 to 14. Fractions 7 to 9 are considered EV‐rich and protein‐ and lipoprotein‐poor and were pooled. (C) Transmission electron microscopy image of the pooled fractions (negative staining), scale bar refers to 1 µm and 200 nm. (D) Particles size distribution in pooled fractions analysed by NTA.
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Extracellular vesicle isolation method from plasma samples. (A) Differential centrifugations followed by filtration and size exclusion chromatography (SEC). (B) Protein concentration (line) on the 14 SEC fractions, combined with western blot analysis using EV‐markers (ALIX and CD9) and Apolipoprotein A1 (APO A1) and B (APO B) as non‐EV markers on fractions 7 to 14. Fractions 7 to 9 are considered EV‐rich and protein‐ and lipoprotein‐poor and were pooled. (C) Transmission electron microscopy image of the pooled fractions (negative staining), scale bar refers to 1 µm and 200 nm. (D) Particles size distribution in pooled fractions analysed by NTA.

Journal: Journal of Extracellular Biology

Article Title: Effect of a 12‐Week Endurance Training Program on Circulating Extracellular Vesicle Proteome in Sedentary Adults With Obesity

doi: 10.1002/jex2.70087

Figure Lengend Snippet: Extracellular vesicle isolation method from plasma samples. (A) Differential centrifugations followed by filtration and size exclusion chromatography (SEC). (B) Protein concentration (line) on the 14 SEC fractions, combined with western blot analysis using EV‐markers (ALIX and CD9) and Apolipoprotein A1 (APO A1) and B (APO B) as non‐EV markers on fractions 7 to 14. Fractions 7 to 9 are considered EV‐rich and protein‐ and lipoprotein‐poor and were pooled. (C) Transmission electron microscopy image of the pooled fractions (negative staining), scale bar refers to 1 µm and 200 nm. (D) Particles size distribution in pooled fractions analysed by NTA.

Article Snippet: The following antibodies were applied: Apolipoprotein (APO) B (1:1000, Santa Cruz, sc‐376818), APO A1 (1:1000, Santa Cruz, sc‐393636), ALG‐2‐interacting protein X (ALIX) (1:500, Cell Signaling Technology (CST), E6P9B), cluster of differentiation (CD) 9 (1:1000, CST, D3H4P), CD63 (1:1000, Santa Cruz, sc‐5275), CD81 (1:1000, CST, D3N2D), tumour susceptibility gene 101 (TSG101) (1:1000, Abcam, Ab30871), calnexin (1:1000, CST, 2433), LC3b (1:1000, Sigma, SAB4200361), TOM20 (1:1000, CST, 42406), peroxisome proliferator‐activated receptor gamma, coactivator 1 alpha (PGC‐1α) (1:1000, Santa Cruz, sc517380), oxidative phosphorylation (OXPHOS) (1:1000, Abcam, Ab110413 ), vascular endothelial growth factor A (VEGF‐A) (1:500, Santa Cruz, sc‐7269), citrate synthase (CS) (1:1000, CST, D7V8B), succinate dehydrogenase A (SDHA) (1:1000, Santa Cruz, sc‐390381), extracellular signal‐regulated kinase (ERK) 1/2 (1:1000, CST, L34F12), p‐ERK 1/2 (1:1000, CST, D13.14.4E), NFκB (1:1000, CST, D14E12), p‐NFκB (1:1000, CST, 93H1), hypoxia inducible factor 1 alpha (HIF‐1α) (1:500, CST, D2U3T), peroxiredoxin (PRDX) 1 (1:1000, CST, D5G12), actin (1:5000, BD Biosciences, 612656) and eukaryotic translation elongation factor 2 (eEF2) (1:1000, CST, 2332).

Techniques: Isolation, Clinical Proteomics, Filtration, Size-exclusion Chromatography, Protein Concentration, Western Blot, Transmission Assay, Electron Microscopy, Negative Staining