Fig 1.
Schematic diagram of the experimental procedure.
The J774A.1 murine macrophage cell line was cultured in EV-free media (EFM) for 24 hours. The cell culture media was then collected and subjected to low-speed centrifugation and filtration to clarify cellular debris and large microvesicles. Aliquots of 36 mL of such pre-cleared conditioned media (CM) were concentrated using UC, C-UC, UF or PEG into 1 mL suspensions, which were then mixed with 2 mL 60% iodixanol individually to generate 3 mL concentrates. DGUC then separated the concentrates into twelve 1 mL fractions. The EV-containing fraction, fraction 7 was subsequently used for further analysis.
Fig 2.
Nanoparticle and protein analysis of concentrates.
Using the four different methods, CM was individually concentrated into 1 mL suspensions, which were then mixed with 2 mL 60% iodixanol. NTA served to determine the number (A) and size (B) of nanoparticles in the EFM, the CM, and the 3 mL concentrate from each method. Protein concentration was quantified by Qubit assay (C). For statistical analysis, a one-way ANOVA followed with Dunnett’s multiple comparison test was used, and C-UC served as the control group. Data are expressed as mean ± SEM from three experiments, *P<0.05; **P<0.01; ***P<0.001.
Fig 3.
Nanoparticle and protein analysis of DGUC fractions.
The 3 mL concentrates from each of the four methods were layered under a discontinuous iodixanol gradient and centrifuged at 100,000 x g. After 18 hours, twelve 1-mL fractions were collected starting from the top. SDS-PAGE served to resolve a 90 μL sample of each fraction for Coomassie staining (A). Protein concentration of each fraction was quantified by Qubit (B). NTA served to analyze the count (C) and mean size (D) of all nanoparticles in each fraction. Data are plotted from three independent experiments as mean ± SEM.
Fig 4.
Western blot analysis of DGUC fractions.
A 37.5 μL sample was taken from each fraction and resolved by SDS-PAGE and probed for EV markers ALIX, CD81, and CD9. Golgi-derived protein GM130 and endoplasmic reticulum-derived marker Calnexin were used as negative controls (A). The density of each fraction was measured by a refractometer (B). Data are plotted from three independent experiments as mean ± SEM.
Fig 5.
Nanoparticle, protein and RNA analysis of the EV containing fraction.
Electron Microscopy of EVs from fraction 7 isolated using different methods with both scale bars representing 100nm (A). Nanoparticles in fraction 7 isolated using different methods were enumerated (B) and sized (C) by NTA. Protein mass was quantified by Qubit assay (D). An equal volume (200 μL) was taken from fraction 7 for miRNA analysis. Levels of microRNAs miR-21, miR-146a and miR-16 were measured relative to the synthetic spike-in UniSp2 by qPCR (E). An equal volume (37.5 μL) and number (3 x109 nanoparticles) from fraction 7 of all four methods were taken and assessed for CD81 and ALIX by western blot. Representative blot images are shown (F). The ratio of nanoparticles count to μg protein was plotted as a relative measurement of purity (G). For statistical analysis, a 1-way ANOVA followed with Dunnett’s multiple comparison test was used, C-UC served as the control group. Data are expressed as mean ± SEM from three experiments, *P<0.05; **P<0.01; ***P<0.001.