There are several checkpoints through the protocol, and before the staining of uEVs. Therefore, it is essential to first verify the amount of protein present in the extract of uEVs. All the research groups that work with extracellular vesicles quantify the protein, as indicated in step 2.1. Supplementary Figure 2 shows a representative 96 well plate containing uEVs fraction in wells 4E, 5E, and 6E. Wells 1A, 2A, and 3A consist of blanks, but if there are no uEVs purified, the wells will take similar color.
After this step, there is a need to verify the presence of uEVs. Supplementary Figure 3 shows a representative result of a polyacrylamide gel, stained with Coomassie blue, to show the amount of proteins present in all the collected fractions, and to perform comparison with other methodologies to isolate uEVs. Among the two different reducing agents, dithiothreitol (DTT) and β-mercaptoethanol, the second one showed better protein yield.
Another important thing is to validate the presence of uEVs using any of the methodologies recommended by the MISEV2018. Supplementary Figure 4 shows a representative result of the enrichment of several proteins such as CD63 and CD9 in the uEVs and the collected fractions used as negative controls. In the uEVs fraction, no visible bands correspond to these proteins, indicating that there is no uEVs isolation.
Supplementary Figure 5 shows a representative result of the uEVs quantification in 12 healthy individuals without any significant or manifested disease, thereby making this method an excellent choice to isolate EVs in homeostatic conditions.
Once isolation of EVs is confirmed, the next step is to prove that the flow cytometer can differentiate between different sizes. Figure 7 shows an example of graphs obtained with the Megamix FSC beads and other commercial beads with different sizes. As shown, r2 value is very close to 1.0, indicating the cytometer's sensitivity to differentiate between 0.1, 0.3, 0.5, and 0.9 µm bead sizes. If the r2 value is less than 0.7, do not use that cytometer for the protocol presented here.
Figure 7: Validation of flow cytometer to discriminate 0.1 – 0.9 µm.The graphs show representative results. (A) Graph of Mean FSC-H VS size of the Megamix beads (black line) and FITC+ beads (red line) the r2 of both beads are close to 1.000. (B) Graph of Mean SSC-H VS size of the Megamix beads (black line) and FITC+ beads (red line) the r2 of both beads are close to 1,000. Please click here to view a larger version of this figure.
It is then essential to verify that all the negative controls are set in the correct position; also, consider avoiding switching staining panels without readjusting cytometer settings since results obtained will be different when antibodies with different fluorochromes are used. Supplementary Figures 6 and Supplementary Figure 7 show two tubes containing the same sample but stained in a different tube using different antibodies; therefore, it is important to verify these details before applying statistics to the results or to perform any calculations. Supplementary Figure 6 is an example of an incorrect analysis, using only the PBS tube and one fluorochrome detector to set the negative and positive gates. On the contrary, Supplementary Figure 7 shows a correct analysis, considering all the negative controls based on the different antibodies with different fluorochromes. These figures endorse the importance of all the controls mentioned here.
The next critical step is to obtain #uEVs/µL (see Figure 8). It is essential to verify that the statistic number will be the same as the generated dot plot; if not, there is a mistake, and the resulting calculations will be wrong.
Figure 8: Analysis strategy to obtain the number of uEVs per microliter.The image shows a representative workflow to obtain the number of uEVs per microliter. (A) Dot plot SSC-H VS FL1-H showing the negative region considering all the tube controls. (B) Dot plot SSC-H VS FL1-H of the CFSE staining tube, highlighted the percentage of staining. (C) The table obtained in the software highlights the percentage and number of uEVs. (D) Calculus to obtain the real number of uEVs present in the sample. Please click here to view a larger version of this figure.
Once the #uEVs/µL is obtained, , one can obtain the number of uEVs/µL for the sizes defined by the Megamix beads by following the procedure shown in Figure 9. It is important to verify the correct statistic number for the generated gate.
Figure 9: Analysis strategy to obtain the number of uEVs per microliter by size.The image shows a representative workflow to obtain the number of uEVs per microliter by size, 0.1 µm. (A) Dot plot SSC-H VS FL1-H shows the negative region considering all the tube controls. (B) Dot plot SSC-H VS FSC-H of the CFSE staining tube, highlights the percentage of staining in the 0.1 µm gate. (C) The table obtained in the software highlights the percentage of 0.1 µm uEVs. (D) Calculus to obtain the real number of 0.1 µm uEVs present in the sample. Please click here to view a larger version of this figure.
Figure 10 is an example of how the #uEVs/µL can be obtained for FL1 that corresponds to CD9. Do the same for all the antibodies and tubes.
Figure 10: Analysis strategy to obtain the number of uEVs per microliter with a marker.The image shows a representative workflow to obtain the number of uEVs per microliter with the marker, CD9+. (A) Dot plot SSC-H VS FL1-H shows the negative region considering all the tube controls. (B) Dot plot SSC-H VS FL1-H of the CD9+ staining tube. (C) The table obtained in the software highlights the percentage of CD9+ uEVs. (D) Calculus to obtain the real number of CD9+ uEVs present in the sample. Please click here to view a larger version of this figure.
An example of the results obtained using this technique is presented in Figure 11.
Figure 11: Example results obtained by the strategies analysis.Representative graphs of the results obtained from 12 healthy individuals. (A) The number of uEVs per microliter. (B) The number of CD37+ uEVs per microliter. (C) The number of CD53+ uEVs per microliter. (D) The number of CD9+ uEVs per microliter. (E) The number of TSPAN33+ uEVs per microliter. (F) The number of ADAM10+ uEVs per microliter. Please click here to view a larger version of this figure.
Supplementary Figure 1: Dot plots and histograms for each fluorochrome used in the example. For this example, three different fluorochromes were used. On the left side, the histogram is shown, and on the right side, the corresponding dot plot is shown. The gates were selected using the autofluorescence tube to obtain the positive gate. (A) Histogram and dot plot for the FL1-H. (B) Histogram and dot plot for the FL2-H. (C) Histogram and dot plot for the FL4-H. Please click here to download this figure.
Supplementary Figure 2: uEVs protein quantification. The image shows a 96-well plate after the incubation with the reagents; each condition is a triplicate. A1 – A3 is the blank. Wells 1 -3 from B to H is the standard solution of bovine serum albumin at different concentrations. B1 – B3: 2 µg/mL. C1 – C3: 1.5 µg/mL. D1 – D3: 1.0 µg/mL. E1 – E3: 0.75 µg/mL. F1 – F3: 0.5 µg/mL. G1 – G3: 0.25 µg/mL. H1 – H3: 0.125 µg/mL. A4 – A6: whole urine. B4 – B6: urine without cells. C4 – C6: supernatant without uEVs. D4 – D6: Urine cells. E4 – E6: EVs diluted 1:10. Please click here to download this figure.
Supplementary Figure 3: Polyacrylamide gel of urine fractions and uEVs isolated by two different methodologies. The image shows a 15% polyacrylamide gel of urine fractions and uEVs isolated with polyethylene glycol (PEG) 8000 or ultracentrifugation. Line 1: protein marker. Line 2: whole urine. Line 3: urine cells. Line 4: Supernatant without uEVs. Line 5 – 7 uEVS isolated with PEG 8000 in PBS. Line 5 without any reducing agent. Line 6 with DTT. Line 7 with β-mercaptoethanol. Line 8 – 10 uEVS isolated with ultracentrifugation. Line 8 without any reducing agent. Line 9 with DTT. Line 10 with β-mercaptoethanol. Please click here to download this figure.
Supplementary Figure 4: Characterization of uEVs. The image shows a Western blot of different markers of uEVs. Line 1: Whole urine. Line 2: Urine without cells. Line 3: Supernatant without uEVs. Line 4: uVEs. The upper panel shows the tetraspanin CD63 40 kDa. The down panel shows CD9 22 kDa. Please click here to download this figure.
Supplementary Figure 5: uEVs protein quantification in healthy individuals. The graph is a representative example of uEVs protein quantification, n = 12 healthy individuals. Please click here to download this figure.
Supplementary Figure 6: Dot plots with an incorrect setting of negative regions. Representative dot plots of different conditions, the negative regions were set using only the PBS tube. (A) to (D) dot plots SSC-H VS FL2-H. (A) PBS tube. (B) PBS + CD37 FITC, CD53 PE, ADAM10 APC tube. (C) autofluorescence tube (D) uEVs + CD37, CD53, ADAM10. (E) to (H) dot plots SSC-H VS FL1-H. (E) PBS tube. (F) PBS + CD9 FITC, TSPAN33 AF647 tube. (G) autofluorescence tube. (H) uEVs + CD9 FITC, TSPAN33 AF647. Please click here to download this figure.
Supplementary Figure 7: Dot plots with a correct setting of negative regions. Representative dot plots of different conditions, the negative regions were set using all the tube controls. (A) to (D) dot plots SSC-H VS FL2-H. (A) PBS tube. (B) PBS + CD37 FITC, CD53 PE, ADAM10 APC tube. (C) autofluorescence tube (D) uEVs + CD37, CD53, ADAM10. (E) to (H) dot plots SSC-H VS FL1-H. (E) PBS tube. (F) PBS + CD9 FITC, TSPAN33 AF647 tube. (G) autofluorescence tube. (H) uEVs + CD9 FITC, TSPAN33 AF647. Please click here to download this figure.
