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Flow cytometric analysis of individual extracellular vesicles
Marca Wauben
Utrecht University Dept. Biochemistry & Cell Biology
Fac. Veterinary Medicine The Netherlands
Extracellular vesicles have changed the way we look at communication in and between
biological systems
EV offer tremendous opportunities for clinical applications ranging from biomarkers for diagnosis or prognosis
to therapeutic application of EV or mimics for drug delivery
Therapeutic application of extracellular vesicles or mimics
Next hurdle to take: Large scale preparation and isolation of well-defined vesicles
Quality control: Quantitative & qualitative analysis Multiparameter analysis of individual vesicles
Vesicle-based biomarkers: A novel class between small molecule and cellular
biomarkers
High potential biomarkers BUT………
Major technical problem is the analysis of specific subsets (rare events) of vesicles in complex body fluids
EVs are heterogeneous in size & composition
Vast majority of EVs released by living cells <200nm in size NO unique markers for different EV-subsets available
Cellular and Molecular Life Sciences 2011; 68(16):2667-88
Great challenge in the EV-field
• Cargo incorporation into EVs is dynamic
Individual EV analysis can discriminate
• Mixed population of EVs
Bulk-based analysis methods e.g. Western-blotting, proteomics, (bead)capture assays, ELISA
To monitor quantitative and qualitative changes in EV-subsets
Great challenge in the EV-field
High throughput analysis at the particle level
Flow cytometer Designed for high throughput analysis of cells applied for EV analysis
EV analysis by flow cytometry
Size:>300 nm Conventional flow cytometry-based analysis
Size: <300 nm High resolution flow cytometry-based analysis
Optimized BD Influx: Nolte-’t Hoen et al. Nanomedicine, 2012 8:712 Van der Vlist et al. Nat. Protocols, 2012 7:1311
Trigger signal
• Uniform parameter to detect all EVs of interest
• Light scatter is useful as a trigger parameter for cells and large EVs
• Small EVs (<300nm) background problems
Scatter-based thresholding Detection of fluorescent nano-sized beads
FSC-based thresholding
100
101
102
103
10410
0
101
102
103
104
thre
sh
old
SS
C
Reduced wide-angle FSC
Nolte-’t Hoen et al. Nanomedicine, 2012 8:712 Van der Vlist et al. Nat. Protocols, 2012 7:1311
100 nm
200 nm
Fluorescence-based thresholding Detection of fluorescent nano-sized beads
100
101
102
103
10410
0
101
102
103
104
threshold
100
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102
103
10410
0
101
102
103
104
threshold
100 nm
beads
200 nm
beads
Reduced wide-angle FSC
Flu
ore
scen
ce
noise
Nolte-’t Hoen et al. Nanomedicine, 2012 8:712 Van der Vlist et al. Nat. Protocols, 2012 7:1311
Vesicle isolation:
Cell culture supernatant
2x 200g
2x 500g
1x 10,000g
1x 100,000g
Pelleted vesicles
Generic label PKH-67
Collection of
density
gradient
fractions
Flow cytometry
(Generic) fluorescent labeling of cell-derived vesicles
Specific protein
labeling
(FL-Ab)
Nolte-’t Hoen et al. Nanomedicine, 2012 8:712 Van der Vlist et al. Nat. Protocols, 2012 7:1311
High-resolution flow cytometric analysis of nano-sized EVs
Reduced wide-angle FSC
100
101
102
103
10410
0
101
102
103
104
PK
H67 F
luore
scence
threshold 0
10000
20000
30000
40000
50000
60000
1,2
6
1,2
4
1,2
2
1,2
0
1,1
8
1,1
5
1,1
1
1,0
8
1,0
7
1,0
6
DC
Num
ber
of events
Density (g/ml)
Quantification Detection
Nolte-’t Hoen et al., Nanomedicine 2012; Van der Vlist/Nolte-’t Hoen et al., Nature Protocols 2012; Van der Vlist et al., J Extracellular Vesicles 2012
MFG-E8 (B-PE)
MH
CII (
AP
C)
100
101
102
103
10410
0
101
102
103
104
100
101
102
103
10410
0
101
102
103
104
vesicles
act-DC
vesicles
non-act-DC
Reduced wide-angle FSC
PH
K67 f
luore
scence
100
101
102
103
104
100
101
102
103
104
1
3
2
Characterization
Proteins
T cell extracellular vesicles
Light scattering
Any flow cytometer can measure something when concentrations are high enough…….
BUT what does the signal mean?
Van der Pol et al. : Theoretical model for vesicle detection by flow cytometry (Single vs. Swarm detection of microparticles and exosomes by flow cytometry) J. Thromb. Haemost. 2012 10:919
Swarm vs. single detection of nano-sized extracellular vesicles by flow cytometry
Regular flow cytometers •Large single EV detection (>300 nm) •Nano-sized EVs detected as ‘swarm’ = multiple vesicles counted as single event
High resolution flow cytometry •Large and nano-sized (~100 nm) single EV detection
Swarm detection influences quantitative and qualitative flow cytometric analysis of nano-
sized EVs
• Regular flow cytometers can be used for swarm detection of nano-sized EVs ‘Bulk-based’ analysis (no information on EV-subsets, no quantitative analysis)
• For high resolution flow cytometry proper concentrations should be used for genuine single nano-sized vesicle-based analysis
-Reproducibility and comparison of results -Development and evaluation of (novel)techniques No gold standard technique available Need for EV-like standards to calibrate and compare Need for sample preparation guidelines Need for sample analysis by several techniques
Need for standardization of high throughput EV-analysis
Need for comprehensive reporting of well-controlled experiments
