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.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

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
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Protocol


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The following instructions are for the isolation of exosomes from pre-cleared plasma. Other sample types can also be used. For details, see the data sheet for the Exosome Isolation Kits, human.