Single Vesicle Flow Cytometry (vFC TM ) JOHN NOLAN, CEO CELLARCUS - - PowerPoint PPT Presentation

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Single Vesicle Flow Cytometry (vFC TM ) JOHN NOLAN, CEO CELLARCUS - - PowerPoint PPT Presentation

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Single Vesicle Flow Cytometry (vFC TM ) JOHN NOLAN, CEO CELLARCUS BIOSCIENCES SELECTBIO EV-BASED DIAGNOSTICS, DELIVERY & THERAPEUTICS FEBRUARY 18, 2020 Workshop Overview MIFlowCyt EV Minimum Information Guidelines for Reporting EV FC

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SLIDE 1

Single Vesicle Flow Cytometry (vFCTM)

JOHN NOLAN, CEO CELLARCUS BIOSCIENCES SELECTBIO EV-BASED DIAGNOSTICS, DELIVERY & THERAPEUTICS FEBRUARY 18, 2020

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SLIDE 2

Workshop Overview

MIFlowCyt EV – Minimum Information Guidelines for Reporting EV FC Methods and Results Vesicle Flow Cytometry (vFCTM) overview Instrument considerations and setup Assay protocol Data analysis protocol Live demo

Beckman Coulter CytoFlex

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SLIDE 3

EV Isolation Methods

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SLIDE 4

MISEV 2018: EV Characterization

EV Quantification

  • Particle number
  • Total protein (lipid, RNA)

Protein composition

  • EV markers
  • Non-EV markers
  • “Small EV” markers

Non-protein components Single vesicle analysis

  • Imaging
  • Impedance
  • Flow cytometry

Protein topology

  • Exofacial
  • Cytoplasmic
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SLIDE 5

Conventional FC of Individual EVs: Pitfalls

Lack of sensitivity, specificity Lack of appropriate calibration Difficulty of standardization Irreproducibility Artifacts!!!

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SLIDE 6

Limitations of Conventional FC for EV Analysis

1. Light scatter as a trigger channel

➢ Depends on size, shape, λ, collection angle, refractive index ➢ Well described by Mie theory ➢ Vesicles scatter 10-100x <beads

2. Coincidence (aka “swarm”)

➢ Depends on [EV], probe volume ➢ Frequency is readily calculated ➢ Can be identified/eliminated by dilution

3. Fluorescence sensitivity and calibration

➢ Required for data/methods sharing ➢ Well-established protocols, reagents ➢ Not widely practiced

SSC Calibur Diameter (um) 1 2 3 4 Intensity 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6 488, 530/30 MFI (log) 1 10 100 1000 Counts 50 100 150 200 250 ST 245 MESF FITC (Blank) 1252 MESF FITC 3789 MESF FITC 9210 MESF FITC 29740 MESF FITC

PS Beads Vesicle

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SLIDE 7

ISEV-ISAC-ISTH EV FC Working Group

  • Need for high throughput EV

analysis

  • Knowledge on biological samples

and EV isolation

  • Development of EV analysis

technology

  • Many end-users
  • Technology development
  • Education
  • Strong connections with Industry
  • Interface with users

SSC on Vascular Biology

  • Development of guidelines for plasma EV isolation
  • Standardization of plasma EV analysis and

functional assays (e.g. coagulation)

  • Many end-users

Marca Wauben, Ger Arkesteijn, Sten Libregts, EstefaniaLozano Andres (Utrecht) Rienk Nieuwland, Edwin van der Pol, Frank Coumans, Leonie de Rond (Amsterdam) John Nolan, Erika Duggan (San Diego) Jennifer Jones, Aizea Morales-Kastresana, Joshua Welsh (Bethesda) Joanne Lannigan, Uta Erdbrugger (Charlottesville) Alain Brisson (Bordeaux) Romaric Lacroix, Stpahne Robert, Fracoise Dignat- George (Marseilles) John Tigges, Ionita Ghiron, Vasilis Toxivaidis (Boston) Bernd Giebel, Andre Goergens, Tobias Tertel (Essen) James Higgenbotham, Bob Coffey (Vanderbilt) An Hendrix, Oliver de Wever (Ghent) Xiaomei Yan (Xiamen)

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SLIDE 8

ISEV-ISAC-ISTH EV FC Working Group

  • Need for high throughput EV

analysis

  • Knowledge on biological samples

and EV isolation

  • Development of EV analysis

technology

  • Many end-users
  • Technology development
  • Education
  • Strong connections with Industry
  • Interface with users

SSC on Vascular Biology

  • Development of guidelines for plasma EV isolation
  • Standardization of plasma EV analysis and

functional assays (e.g. coagulation)

  • Many end-users
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SLIDE 9

Reproducibility Reproducibility Confirm single EV detection Standardization Advanced standardization Reproducibility Reproducibility

Standardized Methods and Results Reporting

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SLIDE 10
  • 3. Assay controls
  • 1. Buffer only

background

  • 2. Buffer with reagents

background

  • 3. Unstained controls

autofluorescence

  • 4. Isotype controls

Fc receptor binding

  • 5. Single stained controls

positivity, compensation

  • 6. Procedural controls

unexpected artifacts

  • 7. Serial dilution

coincidence

  • 8. Detergent-treatment

vesicle lability

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SLIDE 11

Workshop Overview

MIFlowCyt EV – Minimum Information Guidelines for Reporting EV FC Methods and Results Vesicle Flow Cytometry (vFCTM) overview Instrument considerations and setup Assay protocol Data analysis protocol Live demo

Beckman Coulter CytoFlex

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SLIDE 12

Vesicle Flow Cytometry (vFC™)

CELLARCUS BIOSCIENCES INC vFluorRed selectively stains membrane- bound particles vFC measures EVs directly in diluted biofluid, or after purification

  • 1. Dilute
  • 2. Stain:
  • 3. Dilute and measure
  • 4. Calibrate and report

mAb Fluorescence Membrane Fluor Membrane Fluor CD81 PE (MESF) Diameter (nm) Diameter (nm)

  • Uses commercially-available flow

cytometers

  • Lab automation-compatible
  • Membrane probe provides specificity
  • Homogeneous assay: no wash steps
  • Measures EVs directly in biofluid:

no isolation/purification required

  • Sensitive and specific detection: vesicle

size to ~70 nm, cargo to >25 molecules

  • Calibrated measurements for inter-lab,

longitudinal, cross-platform comparisons Fluorescence- triggered flow cytometry

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SLIDE 13 Diameter (nm) 100 200 300 400 500 600 Nanoparticle Concentration (nps/mL) 5e+9 1e+10 2e+10 2e+10 3e+10 3e+10

NTA

Diameter (nm)

vFC™: Standards for EV Analysis

PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1607 PE+: 42063 (97.84%) Pos MFI: 1624

vCalTM RBC EVs staining with PE- anti-CD235ab vCalTM mAb capture beads stained with PE- anti-CD41 vCalTM MESF calibration beads Lipo100TM Vesicle Size Standard

Molecules PE CD41 [HIP8] PE PE-H Count 10 1 10 2 10 3 10 4 38 76 113 151

PE Fluorescence (MESF) PE Fluorescence (MESF) Diameter (nm)

Measurement Standard Uses Data Vesicle size Lipo100TM : synthetic vesicle, extruded through nanopore filters, extensively characterized Calibrate VFC measurements, Immunofluorescence negative control Fluorescence intensity vCalTM MESF beads: Polymer beads (800 nm) with calibrated levels of fluorescence Calibrate fluorescence (MESF units) Enable cross-platform fluorescence measurements Antibody binding vCalTM mAb binding beads: Polymer beads (800 nm) with calibrated mAb capture capacity Qualify antibody conjugates, Calibrate antibody binding, Enable cross-platform measurements Cell-derived EVs EVs prepared from specific cells types expressing characteristic cargo Cargo expression positive control, size and concentration standard, enable cross- platform measurements

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SLIDE 14

vFCTM: Getting Started

Instrument evaluation, configuration and set up

  • Protocol 0 – Essential standards to qualify and calibrate an instrument

Sample preparation and staining

  • Protocol 1 – Sample serial dilutions to establish assay dynamic range, EV

concentration, lack of coincidence/swarm

  • Protocol 2 – EV counting, sizing and cargo analysis

Data processing and analysis

  • Gating
  • Diameter estimation
  • Immunofluorescence calibration
  • Batch export of data to spreadsheet, summary and plots to PPT/PDF
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SLIDE 15

Instrument performance

vCalTM nanoRainbow beads

  • 0.5 nm multifluorophore, multipeak beads
  • Cross calibrated vs MESF, vesicle standards
  • Fixed concentration: counting standard

Protocol and template evaluates:

  • Laser alignment
  • Vesicle detection performance
  • Immunofluorescence calibration
  • Volumetric measurement

CONFIDENTIAL CELLARCUS BIOSCIENCES INC Beckman Coulter CytoFlex

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SLIDE 16

vCalTM nanoRainbow beads

MESF Cross Calibration

FITC MESF Bead 1: 98 Bead 2: 258 Bead 3: 987 Bead 4: 3730 PE MESF Bead 1: 10 Bead 2: 62 Bead 3: 169 Bead 4: 923 Bangs Quantum FITC BD QuantiBrite PE

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SLIDE 17

vFCTM: Getting Started

Instrument evaluation, configuration and set up

  • Protocol 0 – Essential standards to qualify and calibrate an instrument

Sample preparation and staining

  • Protocol 1 – Sample serial dilutions to establish assay dynamic range, EV

concentration, lack of coincidence/swarm

  • Protocol 2 – EV counting, sizing and cargo analysis

Data processing and analysis

  • Gating
  • Diameter estimation
  • Immunofluorescence calibration
  • Batch export of data to spreadsheet, summary and plots to PPT/PDF
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SLIDE 18

CONFIDENTIAL CELLARCUS BIOSCIENCES INC

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SLIDE 19

vFCTM: Getting Started

Instrument evaluation, configuration and set up

  • Protocol 0 – Essential standards to qualify and calibrate an instrument

Sample preparation and staining

  • Protocol 1 – Sample serial dilutions to establish assay dynamic range, EV

concentration, lack of coincidence/swarm

  • Protocol 2 – EV counting, sizing and cargo analysis

Data processing and analysis

  • Gating
  • Diameter estimation
  • Immunofluorescence calibration
  • Batch export of data to spreadsheet, summary and plots to PPT/PDF
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SLIDE 20

CONFIDENTIAL CELLARCUS BIOSCIENCES INC

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SLIDE 21

vFC™: Controls

Dilution Series Establishes dynamic range and allows assessment of coincidence (“swarm”) artifact (multiple EVs). Detergent treatment Detergent solubilizes EV and other vesicles, confirming their vesicular nature

Violet SSC-H Count

10 10 1 10 2 10 3 10 4 10 5 10 6 10 7 300 600 900 1200 1x Lipo100

Diameter (nm) Count

100.0 200.0 300.0 400.0 500.0 600.0 1000 2000 3000 4000

Detergent

HEKB1020

Diameter (nm) Count

100.0 200.0 300.0 400.0 500.0 600.0 102 204 306 408 1:10 1:20 1:40 1:80 1:160 1:320

Violet SSC-A Count

10 1 10 2 10 3 10 4 10 5 10 6 10 7 124 249 373 497
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SLIDE 22

vFC™: EV Size and Light Scatter

CONFIDENTIAL CELLARCUS BIOSCIENCES INC EV Size Calibration

690/50b PE-Cy5-A Count

10 3 10 4 10 5 10 6 125 250 375 500

Membrane Fluor

Diameter (nm) 100 200 300 400 500 600 Nanoparticle Concentration (nps/mL) 5e+9 1e+10 2e+10 2e+10 3e+10 3e+10

Diameter (nm)

Surface Area, NTA (nm 2) 2e+4 4e+4 6e+4 8e+4 1e+5 Di-8-ANEPPS Fluorescence (Chnl #) 20 25 30 35 40 45 50

Membrane Fluor Surface Area (nm2)

Lipo100TM Lipo100TM is characterized by several orthogonal methods (NTA, RPS EM) and serves as a vesicle size standard

NTA

EV Light Scatter EV light scatter is a complex function of particle size and refractive index. Light scatter is different for EVs from different sources, likely indicating differences in refractive index due to differences in EV cargo RBC EVs PLT EVs Size Size v Scatter Scatter

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SLIDE 23

EV Standards: Blood cell-derived EVs

Liposome No antigens RBC EVs PLT EVs No mAb PE-Annexin V PE-anti-CD235ab PE-anti-CD41

PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 10 PE+: 68 (1.04%) Pos MFI: 942 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1088 PE+: 43471 (98.27%) Pos MFI: 1099 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1 PE+: 149 (0.60%) Pos MFI: 560 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 2 PE+: 71 (0.20%) Pos MFI: 537 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 4 PE+: 12 (0.03%) Pos MFI: 254 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 142 PE+: 14855 (33.08%) Pos MFI: 410 PE-A Diameter (nm) 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1612 PE+: 41856 (98.02%) Pos MFI: 1631 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 2 PE+: 71 (0.20%) Pos MFI: 537 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 7 PE+: 10 (0.01%) Pos MFI: 454 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1065 PE+: 73456 (90.23%) Pos MFI: 1162 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 7 PE+: 123 (0.19%) Pos MFI: 1444 PE-A Diameter (nm) 10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 802 PE+: 59340 (96.18%) Pos MFI: 827
  • CD235
  • AnnV
  • CD41
  • AnnV
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SLIDE 24

vFC™: EV Immunofluorescence

ms IgG PE (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 4 PE+: 111 (0.24%) Pos MFI: 439

Lipo100TM ms IgG PE Negative controls Positive Controls Lipo100 bears no antigen, and provides a reference for background and threshold for positivity Depending on the goals of the measurement, immunofluorescence can be expressed as: Isotype control tests for presence of Fc- mediated IgG binding

CD9 PE (MESF) Diameter (nm)

10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 2063 PE+: 70653 (93.90%) Pos MFI: 2166

CD9 PE (MESF) Diameter (nm)

10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 2 PE+: 45 (0.13%) Pos MFI: 3381

CD9 PE (MESF) Count

10
  • 1
10 2 10 3 10 4 10 5 109 218 327 436

CD81 PE (MESF) Diameter (nm)

10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 255 PE+: 79489 (51.23%) Pos MFI: 635

CD81 PE (MESF) Count

10
  • 1
10 2 10 3 10 4 10 5 109 218 327 436

Cell-derived EVs with characterized expression of specific markers Examples: CD9 on PLT EVs CD81 on 293T EVs

ms IgG PE (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 9 PE+: 95 (0.38%) Pos MFI: 476

Analysis

PE-A Diameter (nm)

10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 138 PE+: 13929 (25.73%) Pos MFI: 466

CD9 PE (MESF) Count

10
  • 1
10 2 10 3 10 4 10 5 137 274 411 548

CD9 PE (MESF)

the number “positive” above a defined threshold

  • r

the population median fluorescence intensity (MFI)

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SLIDE 25

vFC™: Antibody Titration

CD41 PE (MESF) Count 10
  • 1
10 2 10 3 10 4 10 5 109 218 327 436 CD41 PE (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1576 PE+: 48339 (89.37%) Pos MFI: 1730 CD9 PE (MESF) Count 10
  • 1
10 2 10 3 10 4 10 5 109 218 327 436 CD9 PE (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 2063 PE+: 70653 (93.90%) Pos MFI: 2166 CD81 PE (MESF) Count 10
  • 1
10 2 10 3 10 4 10 5 109 218 327 436 CD81 PE (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 255 PE+: 79489 (51.23%) Pos MFI: 635 anti-TS PE mix (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1853 PE+: 55766 (91.58%) Pos MFI: 1984 anti-TS PE mix (MESF) Count 10
  • 1
10 2 10 3 10 4 10 5 109 218 327 436 anti-TS PE mix (MESF) Count 10
  • 1
10 2 10 3 10 4 10 5 384 768 1152 1536 anti-TS PE mix (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 486 PE+: 123793 (66.05%) Pos MFI: 862

PLT EVs PLT EVs PLT EVs 293T EVs 293T EVs

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SLIDE 26

vFCTM: EV Analysis Assay kit

Instrument evaluation, configuration and set up

  • Protocol 0 – Essential standards to qualify and calibrate an instrument

Sample preparation and staining

  • Protocol 1 – Sample serial dilutions to establish assay dynamic range, EV

concentration, lack of coincidence/swarm

  • Protocol 2 – EV counting, sizing and cargo analysis

Data processing and analysis

  • Gating
  • Diameter estimation
  • Immunofluorescence calibration
  • Batch export of data to spreadsheet, summary and plots to PPT/PDF
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SLIDE 27

CONFIDENTIAL CELLARCUS BIOSCIENCES INC

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SLIDE 28

CONFIDENTIAL CELLARCUS BIOSCIENCES INC

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SLIDE 29

Summary

Vesicle Flow Cytometry (vFCTM) provides:

  • Selective fluorescence detection of membrane particles
  • Surface area-based size estimates
  • Quantitative cargo immunofluorescence
  • Calibrators and standards, controls
  • Standardized sample preparation and data analysis protocols
  • Unambiguous data interpretation and cross lab comparisons

CONFIDENTIAL CELLARCUS BIOSCIENCES INC

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SLIDE 30

Demo and discussion

CONFIDENTIAL CELLARCUS BIOSCIENCES INC

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SLIDE 31

vFCTM: Immunofluorescence

Diameter (nm) Count

0 100.0 300.0 500.0 1500 3000 4500 6000 Lipo100 +vFRed

Diameter (nm) Count

0 100.0 300.0 500.0 308 616 924 1232

Diameter (nm) Count

0 100.0 300.0 500.0 1500 3000 4500 6000 PLT EVs +vFRed

Diameter (nm) Count

0 100.0 300.0 500.0 1553 3106 4659 6212

PE-A Diameter (nm)

10

1 10 2 10 3 10 4 10 5 10 6

100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 10 PE+: 203 (0.69%) Pos MFI: 359

PE-A Count

10 1 10 2 10 3 10 4 10 5 10 6 10 7 100 200 300 400

PE-A Diameter (nm)

10

1 10 2 10 3 10 4 10 5 10 6

100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 12 PE+: 513 (0.92%) Pos MFI: 351

PE-A Count

10 1 10 2 10 3 10 4 10 5 10 6 10 7 100 200 300 400

PE-A Diameter (nm)

10

1 10 2 10 3 10 4 10 5 10 6

100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 15 PE+: 3890 (16.59%) Pos MFI: 336

PE-A Diameter (nm)

10

1 10 2 10 3 10 4 10 5 10 6

100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1512 PE+: 14793 (78.85%) Pos MFI: 1947

Size No mAb + mAb

Lipo100 PLT EVs

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SLIDE 32

Diameter (nm) Count

100.0 300.0 500.0 239 479 718 957

Diameter (nm) Violet SSC-H

200 400 600 8001000 10 10 1 10 2 10 3 10 4 10 5 10 6 10 7

Violet SSC-A Count

  • 10
10 1 10 2 10 3 10 4 10 5 10 6 10 7 72 143 215 287

Diameter (nm) Violet SSC-A

200 400 600 800 1000
  • 10
2 10 3 10 4 10 5 10 6 10 7

FITC-A Diameter (nm)

10
  • 1
10 3 10 4 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 FITC MFI: 59 FITC+: 85 (0.39%) Pos MFI: 18558

CFSE (FITC MESF) Diameter (nm)

10
  • 1
10 3 10 4 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 FITC MFI: 208 FITC+: 7770 (25.64%) Pos MFI: 4899

Diameter (nm) Violet SSC-H

200 400 600 8001000 10 10 1 10 2 10 3 10 4 10 5 10 6 10 7

CFSE+

Diameter (nm) Violet SSC-H

200 400 600 8001000 10 10 1 10 2 10 3 10 4 10 5 10 6 10 7

TS PE mix (MESF) Diameter (nm)

10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 122 PE+: 10207 (35.67%) Pos MFI: 2318

PE-A FITC-A

10
  • 1
10 3 10 4 10 5 10
  • 1
10 3 10 4 10 6 10 7

13.89% 50.86% 30.37% 4.88%

TS+

vFC of Plasma EVs

CFDA-SE: Esterase-based staining of EV volume Human Plasma (ISTH) Citrated, spun 2x @2500xg, 15 min Freeze aliquots Anti-TS PE mix: CD9 PE CD63 PE CD81 PE

PE-A Diameter (nm)

10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 13 PE+: 13 (0.06%) Pos MFI: 4306

Plasma vFC protocol:

  • 1. Dilute
  • 2. Stain: membrane

probe, volume marker, surface marker

TS PE mix (MESF) CSFE (MESF)

Lipoproteins have higher refractive indices

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SLIDE 33 Diameter (nm) Count 100.0 300.0 500.0 239 479 718 957 Diameter (nm) Violet SSC-A 200 400 600 800 1000
  • 10
2 10 3 10 4 10 5 10 6 10 7 Violet SSC-A Count
  • 10
10 1 10 2 10 3 10 4 10 5 10 6 10 7 72 143 215 287 PE-A Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 24 PE+: 119 (0.39%) Pos MFI: 7133 CFSE (FITC MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 FITC MFI: 208 FITC+: 7770 (25.64%) Pos MFI: 4899 CD235ab PE (MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 70 PE+: 3585 (14.94%) Pos MFI: 672 CFSE (FITC MESF) Diameter (nm) 10
  • 1
10 3 10 4 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 FITC MFI: 261 FITC+: 8420 (29.42%) Pos MFI: 3630 PE-A Diameter (nm) 10
  • 1
10 3 10 4 10 5 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 13 PE+: 13 (0.06%) Pos MFI: 4306 FITC-A Diameter (nm) 10
  • 1
10 3 10 4 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 FITC MFI: 59 FITC+: 85 (0.39%) Pos MFI: 18558 PB450-A FITC-A
  • 10
3 10 4 10 5 10 6 10 7 10
  • 1
10 3 10 4 10 6 10 7 22.85% 38.43% 32.96% 5.76% PC7-A FITC-A
  • 10
2 10 3 10 4 10 5 10 6 10 7 10
  • 1
10 3 10 4 10 6 10 7 7.69% 31.02% 51.54% 9.74% PE-A FITC-A 10
  • 1
10 3 10 4 10 5 10
  • 1
10 3 10 4 10 6 10 7 6.85% 54.43% 21.98% 16.74% Diameter (nm) Violet SSC-A 200 400 600 800 1000
  • 10
2 10 3 10 4 10 5 10 6 10 7 Diameter (nm) Violet SSC-A 200 400 600 800 1000
  • 10
2 10 3 10 4 10 5 10 6 10 7 PB450-A Diameter (nm)
  • 10
3 10 4 10 5 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 BV421+ MFI: 29 VB421+:25 (0.12%) Pos MFI: 4058 PB450-A Diameter (nm)
  • 10
3 10 4 10 5 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 BV421+ MFI: 2 VB421+:6 (0.02%) Pos MFI: 3710 CD41 BV421 Diameter (nm)
  • 10
3 10 4 10 5 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 BV421+ MFI: 834 VB421+:7036 (29.33%) Pos MFI: 9347 Diameter (nm) Violet SSC-A 200 400 600 800 1000
  • 10
2 10 3 10 4 10 5 10 6 10 7 PC7-A Diameter (nm)
  • 10
2 10 3 10 4 10 5 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 PECy7 MFI: 24 PECy7+: 2 (0.01%) Pos MFI: 2388 PC7-A Diameter (nm)
  • 10
2 10 3 10 4 10 5 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 PECy7 MFI: 101 PECy7+: 3 (0.01%) Pos MFI: 3737 AnnV PECy7 Diameter (nm)
  • 10
2 10 3 10 4 10 5 10 6 10 7 100.0 200.0 300.0 400.0 500.0 600.0 PECy7 MFI: 354 PECy7+: 5992 (24.98%) Pos MFI: 12230 Diameter (nm) Violet SSC-A 200 400 600 800 1000
  • 10
2 10 3 10 4 10 5 10 6 10 7 Diameter (nm) Violet SSC-A 200 400 600 800 1000
  • 10
2 10 3 10 4 10 5 10 6 10 7 PC7-A PB450-A
  • 10
2 10 3 10 4 10 5 10 6 10 7
  • 10
3 10 3 10 4 10 5 10 6 10 7 14.93% 55.45% 25.83% 3.78%

CFSE+ CD235+ CD41+ AnnV+ CFSE: Esterase/ volume marker CD235 PE: Glycophorin (RBCs) CD41 BV421: α2 integrin (PLTs) AnnV PECy7: Exposed phosphatidyl- serine

CFSE (MESF) CD235 PE (MESF) CFSE (MESF) CFSE (MESF) CD41 BV421 CD41 BV421 AnnV PECy7 AnnV PECy7

vFC of Plasma EV Subtypes

Plasma vFC protocol:

  • 1. Dilute
  • 2. Stain: membrane

probe, volume marker, surface marker cocktail vFRed: Membrane marker

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SLIDE 34

Questions?

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SLIDE 35

Multicolor EV panel development

CD41 [HIP8] PE PE-H Count

10 1 10 2 10 3 10 4 38 76 113 151

PE Intensity (MESF) Anti-CD41 PE

CD41 [HIP8] APC APC-H Count

10 2 10 3 10 4 37 74 111 148

APC Intensity (MESF) Anti-CD235 APC

CD41 [HIP8] APC Cy7 APC-A750-H Count

10 2 10 3 10 4 42 84 125 167

APC-Cy7 Intensity (MFI) Anti-CD41 APC-Cy7

APC-A Diameter (nm)

  • 10

10

1

10

2

10

3

10

4

10

5

100.0 200.0 300.0 400.0 500.0 600.0 APC MFI: 1429 APC+: 30236 (39.47%) Pos MFI: 2128

APC-A750-A Diameter (nm)

10 10 1 10 2 10 3 10 4 100.0 200.0 300.0 400.0 500.0 600.0

APCCy7+ MFI :491 APCCy7+: 8.82% Pos MFI: 2510

PE-A Diameter (nm)

100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 1612 PE+: 41856 (98.02%) Pos MFI: 1631

PE-A Diameter (nm)

10 10 1 10 2 10 3 100.0 200.0 300.0 400.0 500.0 600.0 PE MFI: 802 PE+: 59340 (96.18%) Pos MFI: 827
  • A. Antibody capture beads

Blank 200 IgG 1200 IgG

  • B. RBC EVs

CD235 PE CD235 APC

  • C. PLT EVs

CD41 PE CD41 APCCy7

PE Fluorescence (MESF) APC Intensity (MESF) APC-Cy7 Intensity (MFI) PE Fluorescence (MESF)

APC not as bright as PE, but still usable for CD235

  • n RBC EVs

APCCy7 is too dim to detect CD41 on PLT EVs