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Isolation and characterization of Extracellular Vesicles

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Carley Ross

on 4 March 2016

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Transcript of Isolation and characterization of Extracellular Vesicles

Flow cytometric detection and sorting of EVs: analysis and characterization of background noise sources that may impact EV fluorescence or scatter detection
by Dr. Carley D. Ross, Dr. Thomas Ramin, Dr. Aliaksandr Kachynski
Beckman Coulter Life Sciences
Cellular and Molecular Life Sciences 2011; 68(16):2667-88.

What are EVs?

Why flow cytometric
sorting with EV?

Analytical ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution
AUC has broad applications for the study of biomacromolecules in a wide range of solvents and over a wide range of solute concentrations

The Astrios EQ is able to detect and sort Extracellular Vesicles
Instrument calibration and standardization requirements are required and are still in flux
Dynamic Light Scatter may be used for a rough estimate of particle size
Analytical Ultra-Centrifuge measures small differences in EV populations by sedimentation velocity with high resolution


AUC Interference
The High Sort had similar EV AUC patterns except a larger, higher concentration particle (group 8).


Groups were separable by their interference and absorbance patterns.

Alignment
P1 Mask, 0 ND in FSC
Trigger on 561-SSC, 0.006% Threshold
Adjust 561-SSC voltage until noise is 5-10K eps
Run Sample and check for swarming


Extracellular Vesicles
Extracellular vesicles (EVs) include exosomes, activation- or apoptosis-induced microvesicles (MVs)/microparticles and apoptotic bodies
Secreted membrane vesicles, heterogeneous in size and composition
Exosomes – 50-100 nm diameter
Flow Cytometric Sorting of Extracellular Vesicle Applications
Size, Complexity and Fluorescence Sensitivity
Separate Unique Populations
Downstream Activation and Lineage Studies
Webber J, Yeung V, Clayton A. (2015) Extracellular vesicles as modulators of the cancer microenvironment. Semin Cell Dev Biol [Epub ahead of print]. [abstract]
Diagnostic or prognostic alterations
Autoimmune, cardiovascular, hematologic, cancer and other diseases
Flow Cytometric
Instrument Requirements for EVs
High Speed Pulse Processing and Electronics
Molecular Therapy (2011) 19 10, 1769–1779. doi:10.1038/mt.2011.164
Sensitive FSC, SSC and Fluorescence Parameters
Understanding instrument and assay capabilities
Can your cytometer
measure and sort EVs?

Forward Scatter
Side Scatter
Fluorescence
Calibration Standards
Polystyrene Beads
Liposomes
Purchased Exosomes
Yeast and Bacteria
Isolation and Staining EVs
Analysis of EVs
Flow Cytometry and
Extracellular Vesicles
Can your system really see EVs?
Isolate and Stain EVs
Methods for bulk and single EV isolation
Lipid staining with DiI, pkH67, 26
Analyze EVs with current and new technologies
Dynamic Light Scatter
Analytical Ultra-centrifugation
Astrios EQ Flow Cytometry Scatter and fluorescence
quantiFlash
Exosomes
Dye aggregates
Benefits
Uniform standardized particles
Easily accessible
Reproducible
Challenges
Flow Cytometric Side Scatter is dependent on index of refraction
Beads have a higher index (1.6) than EV
Benefits
Biological
Index of refraction 1.45
Reproducible
Challenges
Particle mass is different than EV
Additional dyes either packaged in the membrane or inside the liposome
Liposomes may be a good control substance for setting up EV
8 peak beads
Used to measure
MESF
Benefits
Biologically relevant
Matches index of refraction, mass and density
Variation in lots
Challenges
Precipitation reagent may cause interactions
Expensive (50 ug/$350)
Best method for determining instrument settings
Experimental Data
How do EVs compare to polystyrene beads?
The lower the index of refraction, the lower the SSC Signal
EVs from experiments show higher SSC measurements due to EV complexity
Experimental Data
Polystyrene
How many molecules of fluorescein are on your EV?
924
2289
76
Detection Threshold
LEDs
Discrete Trigger and Signal Pulses
Without intrinsic CV spreading from beads
Measures background without lasers, stream
Coarse adjustment
Fine Adjustment
LED Pulse
Set voltage to EV similar voltage on all fluorescent channels
Trigger on 355-620
Measure discrete pulses
Compare to MESF of 8 peaks
EVs and Flow Cytometry
Serial Ultra-centrifugation
Precipitation
Flow Sorting
EV Staining
CD63 Capture Beads
Dynamic Light Scatter
Analytical Ultra-centrifugation
Dynamic Light Scattering (DLS) works by measuring the intensity of light scattered by the molecules in the sample as a function of time.
By measuring the time scale of light intensity fluctuations, DLS can provide information regarding the average size, size distribution, and polydispersity of molecules and particles in solution
Flow Cytometric pkh26 Sorting
Beckman Coulter Proteome Lab XL-A/I
Sedimentation Velocity
determine sedimentation velocity, aggregation, mass and conformation
distribution of sizes in samples which contain a very broad range of sizes
Utilize intrinsic and fluorochome absorbance and index of refraction changes
Determine extracellular vesicle preparation constituents, dye aggregation, lipid contamination, sedimentation velocity and modeled radius

AUC and EV
Measure the change in index of refraction between the solvent and the sample-solvent in a 2 sector sedimentation velocity experiment
Similar to Side Scatter on a flow cytometer, detects all particle populations within the sample as long as the concentration is 0.5 ug/ml
Fast measurements, sensitive to lower concentrations
AUC Absorbance
Measures 190-800 nm absorbance of the population at a given radius location
Xenon Flash lamp with up to 3 unique absorbances taken per cell per scan
Similar to a high-tech spectrophotometer, measures absorbance of sedimenting populations

21 nm
58 nm
104 nm
Polystyrene Bead Sedimentation Velocity
Sedimentation velocity separates particles with different diameters
Polystyrene particles have different density, frictional ratio and mass than EVs
Concentration
Sedimentation Velocity
Estimated
Mass, Radius
Liposomes, 100 nm
Discrete Population
Sedimentation Velocity Less than Beads
Higher Frictional Ratio
Frictional Ratio Higher = Liposome sedimentation with shape distortion "Bubble moving through Liquid"
f/f0=Higher number more aspherical during sedimentation
Purchased Exosomes
MCF7
Interference
Mouse Cell Cancer Line
3,7,12, 15, 25, 50 K RPM
12-15 hrs
Absorbance
AUC Measurement
208, 260, 280 nm
Speed
Low S Value
Aggregates
Buffer, Controls Required
High F ratio
pkh 26 Dye Only
550 nm absorbance
Small Particles
Interference for pkh26, dye only
Some detectable species
Low concentration
Unstained EV, 550 nm absorbance
HeLa EV Unstained Interference
High concentration
Multiple Populations Visible
Some residual absorbance in unstained sample on 550 nm
Low Sort
Absorbance Overlay
Clustered populations
Clustered species and composition for Low Sort
Low Sort HeLa EV Interference
High Sort HeLa EV Interference
High Sort
Absorbance and interference overlay
Cluster into groups
Clustered species and composition of High Sort
Controls
Absorbance
Interference
HeLa EV Analysis
Protein Assay
Drop Drive Amplitude
Swarming
Background
Coincidence events
Two small particles are counted as one
Concentration curve is an effective control
Controls
Sheath Only
Sample Buffer only
Sample and Buffer
81 nm
200 nm
200 nm Post-Sort Purity
Post-Sort
Polystyrene on DLS

80 nm

200 nm

Liposome Stain Controls
Dye Aggregates
Control: Stain no EV
Sort Regions
Control: Unstained EV
pkh67 Stained HeLa EV
Flow Sorting Challenges
Sort speed dependent on electronics and drop drive
70 um tip, 99 KHz drop drive, 70K EPS
Droplet volume to EV is large
At 40K EPS, every other drop has EV
Required to concentrate sample
Sort Purity difficult to determine with buffer and instrument noise (need secondary confirmation)
Serial Ultra-centrifugation
Human Serum
Cell Culture
20 min RT
30 min at 900 x g
Beckman Allegra X-15R
+
= volume PBS
Collect Serum
Place in 50 ml Tube
Beckman Optima-XPN
12K x G 45 min-2 hr
Remove supernatant
Beckman Optima-XPN
100K x g EPS 70 min-4 hr
Create Exosome-Depleted Media
1. Make 20% FBS-Media for cell type
2. Place 25 ml into ultra-centrifuge tubes
3. Place in 70 Ti Rotor (other rotors may differ, match k-factor)
4. Spin for 18 hours at 100K x g.
5. Remove supernatant without disturbing the pellet.
6. Filter through 0.2 um bottle-top filter and store.
7. Add 20% Exosome-Depleted to 0% Media.
Cell culture in log-phase
Rinse with PBS 2X
Exosome-depleted media
Wait 24-48 hrs
30 min at 900 x g
Beckman Allegra X-15R
Beckman Optima-XPN
12K x G 45 min-2 hr
Remove supernatant
Beckman Optima-XPN
100K x g EPS 70 min-4 hr
Remove supernatant
Human Serum
Cell Culture
20 min RT
30 min at 900 x g
Beckman Allegra X-15R
+
200 ul Reagent for 1 mL $10 per sample
Collect Serum
Place in 50 ml Tube
Remove supernatant
Create Exosome-Free Media
1. Make 20% FBS-Media for cell type
2. Place 25 ml into ultra-centrifuge tubes
3. Place in 70 Ti Rotor (other rotors may differ, match k-factor)
4. Spin for 18 hours at 100K x g.
5. Remove supernatant without disturbing the pellet.
6. Filter through 0.2 um bottle-top filter and store.
7. Add 20% Exosome-Free to 0% Media.
Cell culture in log-phase
Rinse with PBS 2X
Exosome-depleted media
Wait 24-48 hrs
30 min at 900 x g
Beckman Allegra X-15R
Remove supernatant
Wait 30 minutes
10 min at 1000 x g
Beckman Allegra X-15R
5 mL Reagent for 10 mL media $32 per sample
Incubate overnight
1 hr at 10k x g
Beckman Allegra X-15R
100 ug/ml
Unstained EV
pkh26 stain only
pkh26 and EV
pkh67 stain only
pkh 26 and 67 and EV
pkh67 and EV
Beckman Optima-XPN
100K x g EPS 70 min-4 hr
+
PBS
Setup Controls
Incubate 5 min
Add equal volume 1% BSA
Beckman Optima-XPN
100K x g EPS 70 min-4 hr
Add 1 ml Cell Suspension to
1 ml Dye Solution
No PBS or Medium
Causes Aggregates
Bulk EV Isolation Methods
Ultra-centrifugation vs Precipitation
VAN DEUN, Jan et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. Journal of Extracellular Vesicles, [S.l.], sep. 2014. Available at: <http://www.journalofextracellularvesicles.net/index.php/jev/article/view/24858>. 
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