Application protocol
Isolation and analysis of human exosomes
Protocol
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.
- 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.
Download the data sheet
View details
References
Additional resources
The following is a list of additional resources that may be of interest:
- Koliha, N. et al. (2015) A multiplex bead platform for the characterization of cell culture–and plasma-derived exosomes. 2015 ISEV, Washington, D.C., USA.
- Koliha, N. (2016) Diversity of extracellular vesicles addressed by immunoaffinity beads. 2016 ISEV, Rotterdam, NL.
- MACSPlex Analysis of plasma-derived extracellular vesicles
- General instructions for the MACSPlex Exosome Kit
- REAfinity Antibodies Brochure: Most specific. More impact.
Materials
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 (# 130-096-343) 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
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
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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