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