Exosomes are membrane-bound extracellular vesicles that are 30-150 nm in diameter and produced by cells. They contain proteins, lipids, and nucleic acids from their cell of origin. The small size of exosomes makes them challenging to isolate and analyze. Common methods for isolating exosomes include differential ultracentrifugation, ultrafiltration, polyethylene glycol precipitation, immunoaffinity capture, microfluidics, and size-exclusion chromatography. Size-exclusion chromatography is considered an ideal method as it produces relatively pure exosome isolates without excessive sample volumes. Exosomal RNA and membranes can be labeled with fluorescent dyes like green fluorescent cell stain and ceramide for visualization and tracking.

2. How exosomes are
Isolated and labelled
at cellular level?

3. Exosomes
• Membrane-bound extracellular vesicles
• Produced in the endosomal compartment of most eukaryotic
cells.
• 30-150 nm in diameter
• Enveloped by a lipid bilayer

4. Discovery
By Stahl and group, Johnstone and group
in 1983
In the maturing mammalian reticulocyte
termed 'exosomes' by Johnstone and
group in 1987.

5. Structure
• A complex of various proteins
including receptors, transcription
factors, enzymes, extracellular
matrix proteins, lipids, nucleic
acids (DNA, mRNA, and miRNA)
• inside and on the surface of the
exosomes constitute their content

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• some proteins specifically arise from cell and
tissue of origin
Example:
• CAMs, integrins, tetraspanins together with
transferrin receptors on the surface of
reticulocytes.

7. Exosome analysis: Small
targets, the big challenge
• The small size of exosomes makes them
challenging to handle and analyze. In flow
cytometry, exosomes can be difficult to distinguish
from cell debris and other small particles, with their
size at or below the limits of some flow cytometers’
sensitivity. Staining with certain dyes or antibodies
can help to distinguish exosomes from other
particles.

8. Methods of Exosome Isolation
1. Differential
Ultracentrifugation
• Separation of particles by sedimentation
• Dependent on their size and density
Differential Ultracentrifugation

9. 2. Ultrafiltration:
1. Use of membranes with specified pore
diameters
2. pre-determined size range
Ultrafiltration

10. • Aqueous PEG solution to wrap exosomes.
• formation of exosome aggregates.
• low-speed centrifugation at 1500× g.
3. Poly-Ethylene Glycol-Based Precipitation:

11. 4. Immunoaffinity Capture
• Separation of specific exosomes.
• Based on the expression of specific surface proteins.
Immunoaffinity capture

12. 5. Microfluidics
• Microfluidic devices to isolate exosomes.
• Based on immunoaffinity, size, and density.

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6. Size-exclusion Chromatography (SEC):
• separates molecules based on their size by
filtration through a gel.
• Gel consists of spherical beads containing pores
of a specific size distribution.
• Molecules of different sizes are included or
excluded from the pores within the matrix.
• Technique uses starting biofluid as a mobile
phase

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• A porous gel filtration polymer as
the stationary phase.
• Adapted into a single-step
isolation system for exosomes
from biological fluids in 2014.
• SEC cannot differentiate between
exosomes and microvesicles of
the same size.
• Where identification of EV
subtype is important to address,
combining SEC with
immunocapture methods is
recommended.

15. Ideal Method for
Exosome Isolation:
• SEC (Size exclusion chromatography).
• low levels of contaminants and co-precipitates, leading to a
relatively homogenous final exosome isolation.
• no requirement of an excessive volume of samples to
isolate exosomes
• Used successfully to isolate, purify and enrich exosomes
from a variety of biological fluids including plasma, serum,
bovine etc..

16. Labeling of Exosomes:
Labeling of exosomal RNA using fluorescent dyes
• Green Fluorescent Cell Stain is a cell-permeant
nucleic acid stain that is selective for RNA.
• virtually nonfluorescent in the absence of nucleic
acids
• the stain exhibits bright green fluorescence when
bound to RNA
• (absorption/emission maxima ~490/530 nm)

17. Labeling of Exosomes:
Labeling of exosomal Membrane using fluorescent dyes
• Ceramide is a red fluorescent cell stain
• (absorption/ emission maxima ~589/617 nm)
• very efficient labeling of the exosomal membrane.
• The application of this fluorescent lipid probe stain also
allows very efficient labeling of the exosomal
membrane.

18. Thank
you