MENU
OCT 01, 2015 9:30 AM PDT

WEBINAR: Extracellular Vesicle Isolation by Flow Cytometric Sorting and Characterization by Analytical Ultra-Centrifugation and Dynamic Light Scatter

Speaker

Abstract

Background:  The extracellular vesicle (EV) research field has dramatically increased in the last five years.  Using a high-speed flow cytometric sorter, EVs may be isolated at high rates such that researchers can differentially separate, isolate and characterize the EVs for downstream analysis.  EVs contaminated with proteins, dye or antibody aggregates of the same size, but different mass, can be characterized based on these physical properties in the analytical ultra-centrifuge. This presentation focuses on characterizing the EVs on an XLA/I AUC and dynamic light scatter isolated by flow cytometric sorting. Methods:  Extracellular vesicles were isolated from a HeLa cell line using serial ultracentrifugation.  HeLa EVs were measured for total protein content and confirmed using CD63 Dyna-beads.  EVs were stained with PKH26 and sorted on the MoFlo Astrios EQ using side scatter and fluorescence.  Instrument performance was tested with polystyrene latex beads ranging from 22- 104 nm, and 100 nm liposomes for minimum detection limits.  Absorbance and interference were used on AUC to measure the EV sedimentation.  DelsaMax (DM) Core, dynamic light scatter (DLS) instruments, were used to measure the particle sizes of EV samples.  Results:  The Astrios EQ was able to distinguish and sort the EVs with some overlap on SSC noise.  100 nm liposomes and 81 nm were visible above noise.  EVs stained with PKH-26 and sorted on SSC and fluorescence with populations above and in the noise.  Sorted fractions were analyzed on the AUC and DM for unstained, stained and aggregate population distributions.  AUC indicated EV populations with varied sizes and dye aggregates and EV size and distribution was measured by the DM with DLS.   Conclusions:  Sorting stained EV populations on a high speed sorter provides for their visualization, separation and characterization.  The AUC effectively separated particles on their sedimentation velocity and clarified issues with dye aggregation vs EV staining.  Addditionally, the DelsaMax allowed for quick analysis of post-sorted populations. The Astrios EQ was able to sort EVs and AUC provided additional analysis for exosome purity.

Learning Objectives:
  • Learn how EVs may be isolated at high rates such that researchers can differentially separate, isolate and characterize the EVs for downstream analysis using a high-speed flow cytometric sorter
  • Learn how EVs contaminated with proteins, dye or antibody aggregates of the same size, but different mass, can be characterized based on these physical properties using the analytical ultracentrifuge
  • This presentation focuses on how to characterize EVs using an XLA/I AUC and dynamic light scatter isolated by flow cytometric sorting

 Beckman Coulter, the stylized logo, Astrios, and DelsaMax are trademarks of Beckman Coulter, Inc.  Beckman Coulter and the stylized logo are registered in the USPTO.


Show Resources
You May Also Like
MAY 11, 2021 10:00 AM PDT
C.E. CREDITS
MAY 11, 2021 10:00 AM PDT
Date: May 11, 2021 Time: 10:00zm PDT Your samples are some of the most valuable assets in the laboratory. After spending countless hours on extraction and preparation, your conclusions could...
SEP 14, 2021 7:00 AM PDT
C.E. CREDITS
SEP 14, 2021 7:00 AM PDT
Date: September 14, 2021 Time: 7am PDT, 10am EDT, 4pm CEST A conventional thermal cycler has long been a commodity product in the lab and end-point PCR techniques can be completed almost wit...
OCT 12, 2021 9:00 AM PDT
C.E. CREDITS
OCT 12, 2021 9:00 AM PDT
Date: October 12, 2021 Time: 9:00am (PDT), 12:00pm (EDT) SCIEX’s next-generation Biologics Explorer software is an innovative platform for the comprehensive and deep characterization o...
OCT 20, 2021 10:00 AM PDT
C.E. CREDITS
OCT 20, 2021 10:00 AM PDT
Date: October 20, 2021 Time:10:00am (PDT), 1:00pm (EDT) As the prevalence of Diabetes continues to rise in many areas across the globe, healthcare providers continue to look for methods that...
AUG 24, 2021 11:00 AM PDT
C.E. CREDITS
AUG 24, 2021 11:00 AM PDT
Date: August 24, 2021 Time: 11:00am (PDT), 1:00pm (EDT) Electron-based dissociation mechanisms have shown great promise for advanced characterization of biomolecules. However, routine adopti...
JUL 15, 2021 9:00 AM PDT
JUL 15, 2021 9:00 AM PDT
Date: July 15, 2021 Time: 9:00am (PDT), 12:00pm (EDT) The Pisces workflow robust, easy-to-use, end-to-end multi-omics solution for highly multiplexed targeted Spatial RNA analysis. VeranomeB...
Loading Comments...
Show Resources