Measurement of extracellular vesicles with tunable resistive pulse sensing.

Step-by-step protocol, also available on protocols.io. (PDF)</p

EV isolation and technical reproducibility following SEC isolation and TRPS measurement.

(A) Western blot comparing prototypical markers and investigational targets in isolated EVs and whole cell lysate from HT1080 cells. (B) Dot plot comparing mean particle size (x-axis) and EV concentration (y-axis) among technical replicates of human plasma. Technical replicates are grouped by color and a correspondingly shaded ellipse. (C) Comparison of sample concentration (y-axis) by technician operating the TRPS instrument. EV concentration detected by technician 1 (median 0.81 x 1011 particles/mL, interquartile range 0.59–1.19 x 1011 particles/mL) and technician 2 (median 0.60 x 1011 particles/mL, interquartile range 0.45–1.10 x 1011 particles/mL, p = 0.606) are displayed separately. A Student’s t-test was used to test for significance.</p

Human sample processing and isolation of extracellular vesicles with size exclusion chromatography.

Step-by-step protocol, also available on protocols.io. (PDF)</p

Full gel/blot images accompanying Fig 1A.

Each column represents visualization of total protein loaded onto the gel (Activation), transferred onto the nitrocellulose (Transfer), or specific target detection (Immunodetection). Bio Rad TGX gels were used for protein separation by molecular weight. These gels contain a trihalo compound which modifies tryptophan residues in protein samples by a covalent modification. When exposed to ultraviolet (UV) excitation, a fluorescence signal is visualized representing total protein in both the gel (Activation) and on the nitrocellulose (Transfer). Chemiluminescent immunodetection is recorded by ChemiDoc Imaging System for each antibody exposure A) CD9 B) CD68 C) CD81 D) CD63 E) GM130 F) GAPDH G) MMP-14 H) MMP-2 I) TGFβ J) TIMP-2. Gel order: Ladder, EVs, whole cell lysate. The yellow box indicates the cropped region included in Fig 1A. (PDF)</p