MNPs!labeling!! AF488! (Citrate).!! 15min · Threshold B525-350 Below threshold Value above comp...

CD31    MNPs  

MNPs  labeling    AF488  15  min    

CD41a    MNPs  

CD63    MNPs  

Blood  collec<on    (Citrate).    

3,000  g  for  15  min  Platelet  Poor  Plasma  

(PPP)  

CD41a-­‐APC    CD63-­‐PE      

PPP  

1   2   3  

Aliquots  at  -­‐80ºC  

CD31-­‐AF  647  CD63-­‐PE      

CD41a-­‐APC  CD31-­‐PE      

Stained   Control  

Isotype  Controls  

Isotype  Controls  

Isotype  Controls  

Magne

<c  se

para<o

n  an

d  washe

s  (x2)  

 An

alysis  by  Flow

 cytom

etry  

t0  

<t0+  1H  

Figure  S1  

                 S1  Figure.  Summary  of  the  protocol  for  capture  and  isola<on  of  EVs.    MNPs  (15  nm)  coupled  to  an5bodies  against  one  of  EVs’  an5gens  were  incubated  with  EVs.    MNPs  were  labeled  by  Zenon  (Fab)  against  the  capture  an5bodies.  Two  other  fluorescent    an5bodies  were  added  to  the  captured  EVs  MNP–EV  complexes  were  separated  on  magne5c    columns  and  analyzed  in  a  flow  cytometer    

Sizing  a(er  ga+ng  on  singlets  

1spin  2spin  

Sizing  without  ga+ng  on  singlets  1spin   2spin  MegaMix-­‐SSC  

SSC

S2  Figure.  Plasma  EVs  a(er  one  centrifuga+on  vs.  two  centrifuga+ons.  A.  Calibra(ng  beads  of  different  size.  B.  Size  distribu(on  of  EVs  a8er  one  spin.    C.  Size  distribu(on  of  EVs  a8er  second  spin.  D.  Ga(ng  of  EVs  captured    with  CD31–MNPs  a8er  one  spin.  E.  Ga(ng  of  EVs  captured    with  CD31–MNPs  a8er  second  spin.     Figure  S2  

A   B   C  

D  

E  

Threshold B525-350

Below threshold

Value above comp control(B525) can not compensate

Part of baseline report showing limit of linearity of Blue-525 PMT

S3 Figure. Fluorescence thresholding and gating strategy A. Dot plot of acquired raw data. B. Gating on single particles by excluding aggregates based on height vs. width plot. The grey areas represent exclusion areas that are not included in further analysis: upper grey part is excluded because of out-of-range events on fluorescence height parameter which are above the linear range defined by cytometer settings based on compensation control (C) and cytometer setup and tracking (CST) procedure (D). Lower grey area is excluded because of events that do not meet criteria for defined threshold (defined threshold is 350 in B525 channel). The vents are recorded as they meet other threshold defined by FSC and represent counting AccuCheck beads.

Figure  S3  

anti-CD31-MNPs (Alexa Fluor 488)

SSC

-A

 B515-A  

B515-W:anti-CD31-MNPs (Alexa Fluor 488)

B51

5-H

:ant

i-CD

31-M

NPs

(A

lexa

Flu

or 4

88)

anti-CD31-MNPs (Alexa Fluor 488)

SSC

-A

100 200 300 400 500 600 700 800 900 1000

66.42

525 155 175 235 355 465

Size(nm)

Con

cent

ratio

n (E

6 pa

rtic

les/

ml)

Averaged FTLA Size/Concentration Red error bars indicate +/-1 standard error of the mean

A B

C D E

S4  Figure.  Size  distribu0ons  of  the  captured  EVs.  A.  Calibra(ng  beads  of  different  size.  B.  Size  distribu(on  of  unfrac(onated  EVs.    C.  Ga(ng  39  strategy  of  EVs  captured  with  CD31–MNPs.  D.  Size  distribu(on  of  the  gated  EVs.  E.  NanoSight  analysis  of  CD31-­‐captured  EVs    

Figure  S4  

CD63 PE CD63 PE CD31 PE

CD41a APC CD31 AlexaFluor647 CD41a APC

CD31 captured EVs CD41 captured EVs CD63 captured EVs A.   B.   C.  

D.   E.   F.  

0.07  %  

0.94  %  

1.00  %  

0.10  %  

0.24  %  

0.18  %  

S5 Figure. Staining of the MNP-captured EVs with isotype control antibodies. Shaded peaks represent staining with isotype antibodies. Numbers shows positive events in isotype control staining compared with specific antibody staining in a typical experiments. A and D: EVs captured with CD31-MNP. B and E: EVs captured with CD41-MNP. C and F : EVs captured with CD63-MNP.  

Figure  S5