Fig 1.
Schematic overview of the EV separation procedures used.
(A) differential centrifugation and ultracentrifugation and (B) exoEasy purification. THP-1 cells were seeded at a density of 1 million cells/ml and exposed to 100 ng/ml LPS for 24 hours in medium containing 2% exosome-depleted FBS. Thereafter, the conditioned cell medium containing the EVs was further processed as described in Materials and Methods.
Fig 2.
Characterization of EVs by scatter-based NTA and protein analysis of EVs.
A representative size distribution profile of particles (solid line) is shown together with the blank medium as background control (dashed line) for (A) DC + UC and (B) exoEasy. Normalized values are shown to the equivalent of EVs isolated from 1 × 106 LPS-stimulated THP-1 cells as described in Materials and Methods. (C) Western blotting analysis of proteins considered as EV-specific markers (TSG101, flottilin-1 and CD9) and the non-EV marker cytochrome c for both separation procedures. The presence or absence of the protein markers in isolated EV fractions was evaluated against blank medium (negative control) and LPS-stimulated THP-1 cell lysate (positive control). Full images of immunoblots are shown in S3 Fig in S1 File.
Table 1.
Characterization of the EVs isolated from THP-1 cells using DC + UC and the exoEasy kit.
Fig 3.
Characterization of EVs by HS-FCM after staining with CFDA-SE.
(A) Dot plots of the side scatter (SSC) or fluorescence (BP530/40) expressed in units of FITC MESF versus forward scatter (FWSC) demonstrating the sensitivity of HS-FCM when measuring 100 and 200 nm fluorescent polystyrene beads. (B) Absence of detector swarming was verified using serially diluted CFDA-SE stained EVs from DC + UC isolated EVs. Additional fluorescence plots can be found in S6 Fig in S1 File. (C) A representative distribution of EV counts in different iodixanol density gradient fractions for both DC + UC (left) and exoEasy (right) isolated EVs and the corresponding blank medium controls. The results are presented as the average ± standard deviation of three technical replicates.
Fig 4.
Evaluation of EV detergent sensitivity by HS-FCM after staining with CFDA-SE.
(A) Dot plots of CFDA-SE fluorescence intensity expressed in units of FITC MESF as a function of forward scatter (FWSC) of EVs and blank medium controls with and without treatment with the detergents 0.5% (w/v) Triton X-100 or 0.5% (w/v) SDS. Representative examples of iodixanol fractions with a density of 1.10 g/ml are presented. Additional fluorescence plots of blank medium controls with and without treatment with the detergents can be found in S7 Fig in S1 File. (B) Relative EV numbers (%) measured during 30 seconds after detergent treatment compared to EVs without detergent (100%) in iodixanol fractions for DC + UC (left) and exoEasy (right) purified EVs. The results are presented as the average ± standard deviation of three biological replicates. For every biological replicate, the average was determined based on three technical replicate measurements. Statistically significant results are indicated by an asterisk (* p-value = 0.0031, ** p-value < 0.001).
Fig 5.
Effect of storage at 4°C or -80°C on EV concentrations.
EV number concentrations were evaluated using (A) scatter-based NTA and (B) fluorescence-triggered HS-FCM on fresh isolates obtained using DC + UC or exoEasy, and after 1 week, 2 weeks and 1 month of storage. A representative experiment out of two is shown. The NTA concentrations were determined in triplicate and results are shown as the average ± standard deviation. For the HS-FCM measurements, the total number of particles in 30 seconds was determined.
Fig 6.
Effect of storage on stability of EV staining fluorescence determined by HS-FCM.
(A) Samples were labeled with CFDA-SE, purified on a density gradient and EV counts (density of 1.10 g/ml) measured for 30 seconds. Relative number of recorded events (%) in 30 seconds measured after different times up to 28 days of storage at 4°C or -80°C in sucrose. Normalization of events was performed between independent experiments (n = 4) and are presented as the average percentage of recorded events compared to the number of events at day 0 with error bars indicating the standard error of the mean. (B) A representative number distribution of the fluorescence intensity (expressed in units of FITC MESF) after one month of storage at -80°C shows that the intensity distribution is comparable between fresh and stored samples. When samples were stored at 4°C, hardly any fluorescent events were found (not shown).