science - all slides emd milliporesigma exosomes webinar ... slides...doeppner, herz, görgens et...
TRANSCRIPT
![Page 1: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/1.jpg)
Brought to you by the Science/AAAS Custom Publishing Office
Participating experts
Analysis of extracellular vesicles including exosomes by imaging flow cytometryJune 22, 2016
Webinar Series
Sponsored by
André Görgens, Ph.D.Essen University HospitalEssen, Germany
![Page 2: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/2.jpg)
Instructions for viewersBrought to you by the Science/AAAS Custom Publishing Office
• To share webinar via social media:
• To see speaker biographies, click: View Bio under speaker name
• To ask a question, click the Ask A Question button under the slide window
• To share webinar via e‐mail:
Analysis of extracellular vesicles including exosomes by imaging flow cytometryJune 22, 2016
Webinar Series
Sponsored by
![Page 3: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/3.jpg)
Analysis of extracellular vesiclesincluding exosomes
by imaging flow cytometry
University Hospital EssenInstitute for Transfusion Medicine
André Görgens, Ph.D
Science/AAAS Technology Webinar Series
June 22, 2016
![Page 4: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/4.jpg)
Human Hematopoiesis
Multi-ParameterFlow Cytometry / Sorting
Görgens et al., Cell Reports 2013Görgens et al., Cell Cycle 2013Görgens et al., Stem Cell Reports 2014
![Page 5: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/5.jpg)
Human Hematopoiesis
Multi-ParameterFlow Cytometry / Sorting
Görgens et al., Cell Reports 2013Görgens et al., Cell Cycle 2013Görgens et al., Stem Cell Reports 2014
Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015Börger, Bremer, Görgens et al., ISBT Science Series 2016
![Page 6: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/6.jpg)
Human Hematopoiesis
Multi-ParameterFlow Cytometry / Sorting
Görgens et al., Cell Reports 2013Görgens et al., Cell Cycle 2013Görgens et al., Stem Cell Reports 2014
Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015Börger, Bremer, Görgens et al., ISBT Science Series 2016
Specific markers andoptimized methodsrequired to resolveEV heterogeneity
![Page 7: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/7.jpg)
Extracellular Vesicles (EVs)
shedding
EVs are secreted by a wide range of cells from different species.EVs can be found in nearly any body fluid.
(Microparticle)
![Page 8: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/8.jpg)
Extracellular Vesicles (EVs)
Putative physiological functions: antigen presentation, intercellular communication, shuttling of RNAs between cells …
shedding(Microparticle)
![Page 9: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/9.jpg)
Extracellular Vesicles (EVs) Heterogeneity
How can EVs/Exosomesbe analyzed?
![Page 10: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/10.jpg)
Methods for EV analysis
Size distributionParticle concentration
• Nanoparticle Tracking Analysis
• Electron Microscopy, Resistive Pulse Sensing, …
![Page 11: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/11.jpg)
Methods for EV analysis
• All methods currently available are limiting, including flow cytometry (size, signal intensity)
• New & improved methods for EV analysis highly desired by the field
Size distributionParticle concentration
• Flow Cytometry
• Nanoparticle Tracking Analysis
• Electron Microscopy, Resistive Pulse Sensing, …
![Page 12: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/12.jpg)
Submicron-Sized Polystyrene Beads as Calibrators?
based on: „Set-Up of the CytoFLEX* for Extracellular Vesicle Measurement”,Prof. Dr. Andreas Spittler, Medizinische Universität Wien (Beckman Coulter White Paper)www.beckmancoulter.de
Biocytex Megamix-Plus SSC/FSC beads
![Page 13: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/13.jpg)
Submicron-Sized Polystyrene Beads as Calibrators?
based on: „Set-Up of the CytoFLEX* for Extracellular Vesicle Measurement”,Prof. Dr. Andreas Spittler, Medizinische Universität Wien (Beckman Coulter White Paper)www.beckmancoulter.de
Biocytex Megamix-Plus SSC/FSC beads
Flow Cytometer
Comparison:
none of these werebuilt for analysis of
submicron particles!
• Beckman Coulter FC500• Beckman Coulter Gallios• Beckman Coulter Cytoflex• ACEA Novocyte 3000• BD Accuri C6• BD FACSAria IIIu• Amnis ImageStreamX MkII
![Page 14: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/14.jpg)
Analysis of Submicron-Sized Beads
![Page 15: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/15.jpg)
Analysis of Submicron-Sized Beads
AmnisImageStreamX Mk II
![Page 16: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/16.jpg)
Imaging Flow Cytometry – Amnis ImageStreamX MkIISubcellular localisationImmunophenotype Morphology
Amnis ImagestreamX MkII
5 lasersup to 12 channels Automatic triggering on all channels
Amnis - part of MilliporeSigma a business of Merck KGaA, Darmstadt, Germany
![Page 17: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/17.jpg)
Imaging Flow Cytometry – Amnis ImageStreamX MkII
Amnis - part of MilliporeSigma a business of Merck KGaA, Darmstadt, Germany
![Page 18: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/18.jpg)
Amnis - part of MilliporeSigma a business of Merck KGaA, Darmstadt, Germany
![Page 19: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/19.jpg)
Analysis of Microparticles Using Imaging Flow Cytometry
![Page 20: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/20.jpg)
Submicron-Sized Polystyrene Beads as Calibrators
![Page 21: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/21.jpg)
Submicron-Sized Polystyrene Beads as Calibrators
Beads EVs?Exosomes?
24 nm beads (Thermo Scientific)BF2BF1 SSC Ch02
![Page 22: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/22.jpg)
HEK293T CD63-eGFP
CD63-eGFP EVs as Biological Calibrator for Flow Cytometry
![Page 23: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/23.jpg)
CD63-eGFP EVs as Biological Calibrator for Flow Cytometry
![Page 24: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/24.jpg)
CD63-eGFP EVs as Biological Calibrator for Flow Cytometry
![Page 25: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/25.jpg)
CD63-eGFP EVs as Biological Calibrator for Flow Cytometry
![Page 26: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/26.jpg)
Lysis Control
0.5 % Nonidet NP-4030 min
![Page 27: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/27.jpg)
Evaluation of Acquisition Parameters
![Page 28: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/28.jpg)
Evaluation of Acquisition Parameters
![Page 29: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/29.jpg)
Optimization of EV Masking
![Page 30: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/30.jpg)
Optimization of EV Masking
![Page 31: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/31.jpg)
Detection of Single Vesicles vs. Swarm Detection
![Page 32: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/32.jpg)
Detection of Single Vesicles vs. Swarm Detection
![Page 33: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/33.jpg)
Summary
• Range of linearity• Analysis parameters• Variability• Comparison of different
machines
• eGFP-positive biological calibrator to optimize imaging flow cytometryfor EV analysis
• Evaluation of acquisition parameters• Optimization of masking• Discrimination of single EV detection from swarm detection
Imaging Flow Cytometry…• … facilitates reliable detection & quantification of single, fluorescent EVs• … absolute quantification (objects/mL sample) of low volume samples (15 µL)• … high sensitivity, clear separation of signal from background/noise• … image-based features to optimize analysis post acquisition
(i.e. exact discrimination of doublets or background)
![Page 34: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/34.jpg)
How can we apply this protocol?
• Evaluation of EV isolation methods• Validation of antibody staining protocols
• Validation of other specific EV probes(fluorescent dyes)
![Page 35: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/35.jpg)
Re-Evaluation of Exosome Isolation Protocols
![Page 36: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/36.jpg)
Re-Evaluation of Exosome Isolation Protocols
57%
40%
7%
100%
76%
![Page 37: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/37.jpg)
EVs+
CD63-PE
EVs+
Isotype-PE
PBS+
CD63-PE
unstained
eGFP
PE
eGFP
PE
1:50 1:25 1:10
Staining of EVs with Antibodies
![Page 38: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/38.jpg)
PE
SS
C
PBS + antibody (buffer controls)
Antibodies – Background Issues
Antibody A
PE
Antibody B
PE
Antibody C
![Page 39: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/39.jpg)
• Optimized protocol for EV analysis includingExosomes using Imaging Flow Cytometry(Görgens et al, in preparation)
• Robust quantification of EV subsets• Can be used with unprocessed samples• Robust multiparametric surface analysis of
single EVs• CD63-eGFP EVs as a biological calibrator/tool
for protocol optimization and validation
Robust high-resolution analysis of EVs subpopulations • with specific, validated probes • in all sample types/unprocessed samples
Summary
BF1 BF2 SSC eGFP PE PE-Dz594 APC
![Page 40: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/40.jpg)
University Hospital EssenUniversity of Duisburg-EssenInstitute for Transfusion MedicineProf. Peter A. Horn
Bernd Giebel lab
Michel BremerFlorian MurkeRita Ferrer TurAnna-Kristin LudwigSören JansenVerena BörgerAikaterini KrikoniSymone VitorianoIoana CosgareaAmina BecicMartina Cremanns
Acknowledgements
Coralie GuérinChantal Boulanger
Sebastian SethChristin ProbstSherree Friend
Peter Rhein
Uta ErdbrueggerJoanne Lannigan
Helmut HanenbergBernhard Singer
Adalbert KrawczykVera Rebmann
Collaborators
![Page 41: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/41.jpg)
Brought to you by the Science/AAAS Custom Publishing Office
To ask a question, click the Ask aQuestionbutton under the slide window
Analysis of extracellular vesicles including exosomes by imaging flow cytometryJune 22, 2016
Webinar Series
Sponsored by
Participating experts
André Görgens, Ph.D.Essen University HospitalEssen, Germany
![Page 42: Science - All slides EMD MilliporeSigma Exosomes webinar ... slides...Doeppner, Herz, Görgens et al., Stem Cells Transl Med 2015 Börger, Bremer, Görgens et al., ISBT Science Series](https://reader034.vdocument.in/reader034/viewer/2022051608/604050fa0cf9c232bc51408a/html5/thumbnails/42.jpg)
Look out for more webinars in the series at:
webinar.sciencemag.org
To provide feedback on this webinar, please e-mailyour comments to [email protected]
Brought to you by the Science/AAAS Custom Publishing Office
For related information on this webinar topic, go to:
www.emdmillipore.com/amnis
Analysis of extracellular vesicles including exosomes by imaging flow cytometryJune 22, 2016
Webinar Series
Sponsored by