SmartSEC-DeLipo™ Advanced sEV Isolation Kit

Figure 2. sEV isolated with SmartSEC-DeLipo^TM is much purer. (A) sEV isolated from 250ul of serum with SmartSEC-DeLipo^TM shows higher expression of EV markers, such as CD9, CD81 and TSG101 than those isolated with SmartSEC^TM single when same amount of EV protein were applied for western blot. When same volume of eluted sEV samples were applied for western blot, EV markers’ expression is about the same for sEV isolated from 250ul of serum with SmartSEC-DeLipo^TM or SmartSEC^TM single. (B) sEV isolated from 250ul of plasma or breast milk with SmartSEC-DeLipo^TM shows higher expression of EV markers, such as CD9, CD81 and TSG101 than those isolated with SmartSEC^TM single when same amount of EV proteins was applied for western blot. (C) fNTA data shows that sEV particle number were decreased in plasma or breast milk sample when isolated with SmartSEC-DeLipo^TM in comparison with that isolated with SmartSEC^TM single. However, when purity was evaluated with EV particle number per same amount of EV protein, sEV isolated with SmartSEC-DeLipo^TM demonstrates higher purity than those isolated with SmartSEC^TM single.

Figure 3. Most of the Lipoproteins are removed in sEV isolated with SmartSEC-DeLipo^TM. (A) ApoB and ApoE lipoproteins’ concentrations were evaluated in serum sample, sEV isolated from serum with SmartSEC^TM single and sEV isolated from serum with SmartSEC-DeLipo^TM by ELISA. SmarSEC^TM single dramatically reduced Lipoproteins in isolated sEV and SmartSEC-DeLipo further deplete the remaining Lipoproteins in yield sEV. (B) Percentage of ApoB and ApoE depletion was calculated based on ApoB and ApoE concentration in sEV isolated with SmartSEC-DeLipo^TM vs. SmartSEC^TM single from serum, plasma and breast milk samples.

Figure 4. sEV isolated with SmartSEC-DeLipo^TM shows typical EV morphology. Transmission electron microscopy (TEM) of sEVs isolated from serum using SmartSEC-DeLipo^TM possess typical EV morphology—intact vesicles with a double layer of membranes.

Figure 5. sEV isolated with SmartSEC-DeLipo^TM is biologically functional to delivery small RNA cargo to the recipient cells. (A) Cy3-labeled control siRNA were loaded without or with sEV isolated from serum with SmartSEC^TM single or SmartSEC-DeLipo^TM using Exo-Fect siRNA/miRNA reagent (EXFT200A-1) and delivered to HEK 293 cells. Cells imaged 36-48 hours post transfection. Shown are the bright-field images (bottom row), and fluorescence images (top row). Both sEV isolated with SmartSEC single or SmartSEC-DeLipo^TM can efficiently transfer Cy3-labeled siRNA to most of the imaged cells. (B) HPRT siRNA were loaded without or with sEV isolated from serum with SmartSEC^TM single or SmartSEC-DeLipo^TM using Exo-Fect siRNA/miRNA reagent (EXFT200A-1) and delivered to HEK 293 cells. Cells were lysed and applied for western blot 48 hours post transfection. Both sEV isolated with SmartSEC^TM single or SmartSEC-DeLipo^TM can efficiently transfer HPRT siRNA to HEK 293 cells and achieve knockdown of the endogenous HPRT expression.

Figure 6. sEV isolated with SmartSEC-DeLipo^TM can be used for small RNA profiling and biomarker discovery. sEV isolated from normal vs. pancreatic cancer patient plasma with SmartSEC-DeLipo^TM were used for sEV RNA isolated using EVery100B-1. Then RNA samples were analyzed by Agilent Bioanalyzer. (A) Electrophoretic spectrums and gel images of the sEV RNA derived from the plasma of pooled normal plasma. Then sEV RNA samples from normal and pancreatic patient sample were reverse transcribed into cDNA using Every 200B-1, followed with Every miRNome Profiler (EVery600B-1) (B) Heat map of normal vs. pancreatic cancer plasma miRNA expression profiler. Representative data normalized to global mean of each sample. Pancreatic cancer patient plasma showed distinct miRNA expression profiles compared to normal plasma. (C) Differential expression of selected miRNAs in normal vs. pancreatic cancer plasma. Data normalized with global mean. Expression of miR-25-3p, miR-19b-3p, miR-451a, miR-486-5p, miR-103a-3p and miR-19a-3p are significantly upregulated, while expression of miR-185-3p, miR-424-5p, miR-140-5p and miR-125a-5p is significantly downregulated in pancreatic cancer plasma compared to normal one.

Figure 7. sEV isolated with SmartSEC-DeLipo^TM can be used for proteomic studies. sEV isolated from normal vs. pancreatic cancer patient plasma with SmartSEC-DeLipo^TM or SmartSEC^TM single were analyzed by mass spectrometry. (A) Heatmap of normal vs. pancreatic cancer plasma sEV proteomic profiler using either SmartSEC^TM single or SmartSEC-DeLipo^TM. Pancreatic cancer patient plasma sEV showed distinct proteome compared to normal plasma. (B) sEV from normal pooled plasma isolated with either SmartSEC^TM or SmartSEC-DeLipo^TM also showed differentiated proteome.