Application protocol

Isolation and analysis of human exosomes

In this application protocol, we describe an effective workflow to isolate and analyze exosomes from human plasma. Exosomes are pre-enriched with the Exosome Isolation Kit using MACS® technology, which enables easy and fast screening of potential exosome surface proteins with the MACSPlex Exosome Kit.

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Protocol

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1. Isolation of exosomes

Isolate exosomes or extracellular vesicles from pre-cleared plasma by positive selection using MicroBeads for tetraspanin proteins CD9, CD63, or CD81. Follow the protocol in the kit data sheet.

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2. Flow cytometry analysis of exosomes

Use the MACSPlex Exosome Kit to capture and stain the isolated exosomes. The cocktail of MACSPlex Capture Beads included in this kit are coupled to antibodies that bind to specific exosomal surface epitopes. Bound exosomes are then stained with APC-conjugated antibodies (against tetraspanins CD9, CD63, and CD81) which leads to the formation of complexes, each consisting of MACSPlex Capture Bead, exosome, and APC-conjugated antibody. Follow the protocol of the kit data sheet.
Analyze these complexes with a flow cytometer based on the fluorescence characteristics of both the MACSPlex Capture Beads and the APC-conjugated antibodies.

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MACSPlex Exosome Kit, human

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Formation of complexes that enable flow cytometry analysis of exosomes. With the MACSPlex Exosome Kit, dye-labeled capture antibody beads bind to surface proteins on exosomes and to APC-conjugated detection antibodies. The fluorescence of the capture beads and detection antibodies are used for flow cytometry analysis.

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Stronger signal intensity from extracellular vesicles isolated with MicroBeads compared to ultracentrifugation. Surface marker profiles of extracellular vesicles isolated from plasma of donor A (top) or B (bottom) by ultracentrifugation or immunomagnetic isolation using CD9, CD63, or CD81 MicroBeads. Data indicate APC median signal intensities of isolated extracellular vesicles incubated with the 39 MACSPlex Exosome Capture Beads and stained with a cocktail of CD9-, CD63-, and CD81-APC antibodies. REA and mlgG1 are isotype control MicroBeads.

References and resources

References

Additional resources

The following is a list of additional resources that may be of interest:

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Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

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Materials

Reagent and instrument requirements

For isolation of exosomes

Exosome Isolation Kit CD9, human (# 130-110-913) or
Exosome Isolation Kit CD63, human (# 130-110-918) or
Exosome Isolation Kit CD81, human (# 130-110-914)
Exosome Starting Kit CD9, human (# 130-111-573) or
Exosome Starting Kit CD63, human (# 130-111-576) or
Exosome Starting Kit CD81, human (# 130-111-575)

μMACS™ Separator (# 130-042-602)
MACS® MultiStand (# 130-042-303)
Centrifuge
1.5 mL tubes
Exosome lysis and elution buffer for direct loading of exosomal proteins on sodium dodecyl sulfate (SDS) gels, e.g., 50 mM Tris-Cl, pH 6.8, with 2% (w/v) SDS, 8% (v/v) glycerin, and 0.005% (w/v) bromophenol blue.
(Optional) EDTA or citrate tubes
(Optional) Phosphate-buffered saline (PBS)

For flow cytometry analysis of exosomes

MACSPlex Exosome Kit, human (# 130-108-813)
MACSQuant® Analyzer 10 (# 130-108-813) or other flow cytometer equipped with blue (488 nm) and red (635 nm) lasers
MACS Chill 96 Rack (# 130-094-459) when using the MACSQuant Analyzer 10
MACSQuant Calibration Beads (#130-093-607) when using the MACSQuant Analyzer 10

Microtiter plate format

Orbital shaker for 96-well plates (frequency 450 rpm)
Vacuum manifold or centrifuge with adapters to accommodate microtiter plates
Multichannel pipettor

Tube format

MACSmix™ Tube Rotator (# 130-090-753) or an orbital shaker for tubes (450 rpm)
Polypropylene or polystyrene reagent tubes
96-well round bottom plate

Good to know

Other sample types

The Exosome Isolation Kits can also be used to isolate exosomes from pre-cleared cell culture supernatant, pre-cleared ascites or urine, and from extracellular vesicle preparations (e.g., ultracentrifugation or density gradient centrifugations). For details, see the kit data sheet.

Choice of Exosome Isolation Kit

After exosome isolation using MicroBeads, the respective epitope is predominantly blocked by MicroBeads
and low signals will be detected on the respective MACSPlex bead type (e.g. CD9, CD63, or CD81). Therefore,
the Exosome Isolation Kit Pan, human, which contains a cocktail of CD9, CD63, and CD81 MicroBeads, is not
recommended to be used prior to the MACSPlex Exosome Kit, human.

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Application protocol

Isolation and analysis of human exosomes

Protocol

Materials