Version 2b Last updated 24 November 2022

ab267479
Exosome Isolation and
Analysis Kit - Flow
Cytometry, Plasma
(CD9/CD81)

For the isolation and analysis of exosome from plasma, urine or cell
culture media.

This product is for research use only and is not intended for
diagnostic use.

Copyright © 2022 Abcam. All rights reserved

Table of Contents

1. Overview 3
2. Protocol Summary 4
3. General guidelines, precautions, and troubleshooting 5
4. Materials Supplied, and Storage and Stability 6
5. Materials Required, Not Supplied 6
6. Reagent Preparation 7
7. Sample Preparation 8
8. Assay Procedure 11
9. Typical Data 13
11. Notes 16

Copyright © 2022 Abcam. All rights reserved

1. Overview

Exosome Isolation and Analysis Kit - Flow Cytometry, Plasma

(CD9/CD81) (ab267479) is a simple immunobead assay for
isolation/detection of exosome, using a bead-bound anti-CD9
capture antibody and a PE conjugated anti-CD81 detection
antibody. The kit provides reproducible results and can be run in
parallel to exosome immunophenotyping.

The kit is intended for the immunoisolation (immunomagnetic or

FACS) and Flow Cytometry analysis of pre-enriched CD9/CD81
human exosomes from biofluids (plasma, serum, urine) or cell culture
media.
2. Protocol Summary

Prepare all reagents and samples as instructed

Add 50 µL resuspended capture beads to round bottom tubes. Add

10-15 µg (up to 100 µL) of exosomes isolated by differential
centrifugation. Mix well and incubate in the dark overnight at RT.

Wash with 1 mL of 1X Assay Buffer.

Collect the beads either on magnetic rack or by centrifugation.

Remove supernatant, taking care to not disturb the microspheres.

Add 5 µL of Primary DetectionAntibody (CD81-PE). Mix well and

incubate in the dark for 60 mins at 4°C. Wash with 1X Assay Buffer.

Collect the beads either on magnetic rack or by centrifugation.

Remove supernatant, taking care to not disturb the microspheres.

Resuspend in 350 µL 1X assay buffer and acquire on a flow

cytometer (store for a maximum of 2 hours in the dark).
3. General guidelines, precautions, and troubleshooting

 Please observe safe laboratory practice and consult the safety

datasheet.
 For general guidelines, precautions, limitations on the use of our
assay kits and general assay troubleshooting tips, particularly for
first time users, please consult our guide:
www.abcam.com/assaykitguidelines
 For typical data produced using the assay, please see the assay
kit datasheet on our website.
4. Materials Supplied, and Storage and Stability

 Store kit at +4°C in the dark immediately upon receipt and

check below for storage for individual components. Kit can be
stored for 1 year from receipt, if components have not been
reconstituted.
 Aliquot components in working volumes before storing at the
recommended temperature.
 Do not freeze.
Storage
Item Quantity conditio
n
6000
CD9+ Capture beads beads/test +4°C
(50 µL/test)
Primary detection antibody: Anti-
5 µL/test +4°C
CD81-PE [Clone M38]
Assay Buffer 10X 10 mL +4°C

5. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to
successfully perform this assay:
 Pre-enriched exosomes by ultra-centrifugation.
 Magnetic Rack; 12-hole, 12x75mm.
 12x75 mm Polystyrene Round Bottom Tubes (cytometer tubes).
 Sterile syringe filter with a 0.45 µm pore.
 Syringe of adequate volume
6. Reagent Preparation

 Equilibrate all reagents to room temperature (18-25°C) prior to

use. Before using the kit, spin tubes and bring down all
components to the bottom of tubes.
 Prepare only as much reagent as is needed on the day of the
experiment.

6.1 CD9+ Capture beads:

Polystyrene micro-particles with Mean Diameter (µm) 6.5±0.2
(CV<5%), having dicrete fluorescence intensity characteristics.
Ready to use.
6.2 Primary detection antibody: Anti-CD81-PE:
Ready to use.
6.3 Assay Buffer 10X:
Dilute contents of the 10X Assay Buffer to 1X (PBS 1% BSA) in
PBS, for use in this assay. Assay buffer 1X can be filtered before
use to avoid microbial contamination of the reagent.
7. Sample Preparation

General sample information:

 The kit allows the detection of isolated exosomes from
differential ultracentrifugation as well as direct detection in the
sample without the need for ultracentrifugation, just with simple
pre-treatment.
7.1 Purification of Exosomes by Differential Ultracentrifugation.
 The kit has been validated for pre-enriched human exosomes
from cell culture and bodily fluids, such as serum/plasma, and
urine, through an ultracentrifugation protocol.
 For reference, after pre-enrichment protocol, exosomes
resuspension at 2.4x109 /50µL concentration will be suitable.
 The principle for exosome purification is the same for cell culture
and bodily fluids, but due to the viscosity of some fluids it is
necessary to dilute them with an equal volume of PBS, before
centrifugations.
Figure 1. Workflow for the exosome pre-enrichment based on differential
ultracentrifugation.

7.2 Sample pretreatment for direct exosome detection on human

plasma.
 The sample pretreatment for direct exosome detection from
plasma is not recommended for detection of exosomes from
any otherbody fluids or cell culture media.
 100 -1000 µL of plasma typically provides enough exosomes for
most standard types of analysis.

Figure 2. Plasma pre-treatment workflow for direct exosome detection.

8. Assay Procedure

8.1 Isolate CD9+ exosomes:

8.1.1 Resuspend capture beads by vortex for approximately 20 s.
8.1.2 Add 50 μL of the capture bead to each 12x75 mm
Polystyrene Round Bottom tube (cytometer tube).
8.1.3 Add between 10-15 µg of exosomes isolated by differential
ultracentrifugation or 100 µl for direct exosomes, previously
prepared according to Section 7, to the appropriate tubes.
Mix the reactions gently by pipetting up and down several
times with a pipette and vortexing for few seconds.
8.1.4 Incubate in the dark overnight at room temperature. DO NOT
STIR.
8.1.5 After overnight incubation wash the sample (bead-bound
exosomes) by adding 1 ml of Assay Buffer 1X.
8.1.6 Collect the Magnetic beads by placing tubes on a magnetic
rack and incubate 5 minutes or by centrifugation at 2,500 x g
for 5 minutes. Remove supernatant from tubes by Hand-
decanting in the case of using the magnetic rack (Fig. 4) or
by aspiration. Take care not to disturb the microspheres, and
make sure not to leave more than 100 µL of supernatant in
the tube.
8.2 Stain exosomes for flow cytometry:
8.2.1 After overnight incubation, add the suggested volume
indicated of the Primary detection antibody (5μL/test of the
supplied antibody) to the bead-bound exosomes tube. Mix
gently by pipetting and/or by tapping. It is advisable to
prepare an additional tube with the appropriate isotype
control or without exosomes, for background determination.
8.2.2 Incubate in the dark 60 min at 2-8ºC, without stirring.
8.2.3 Wash the sample (bead-bound exosomes) by adding 1 mL of
Assay Buffer 1X.
8.2.4 Collect the Magnetic beads by placing tubes on a magnetic
rack and incubate 5 min or by centrifugation at 2,500 x g for
5 min. Remove supernatant from tubes by hand-decanting in
the case of using a magnetic rack or by aspiration.
 Note: Take care not to disturb the microspheres, and make
sure not to leave more than 100 μL of supernatant in the
tube.
8.2.5 Resuspend the sample in 350 μL Assay Buffer 1X and Acquire
on a flow cytometer or store in the dark max up to 2 hours at
2-8ºC, until the analysis is carried out.
8.3 Assay Acquisition:
 An adequate gating strategy FSC / SSC and PerCP/APC, PerCP-
Cy5/APC or PerCP-Cy5.5/APC, helps to bead population
identification and discrimination of doublets on flow cytometer.
8.3.1 Gate on the single population(s) on a Forward Scatter vs.
Side Scatter plot in linear scale (Figure 3A).
8.3.2 Gate on the single population(s) on a FL3 vs. FL4 channel
(bead auto fluorescence) in logarithmic scale (Figure 3B).
8.3.3 Using the FL2 channels, determine whether or not any bead
populations tested “positive” for the exosome.
 Note: A positive bead will produce a fluorescent peak in the FL2
channel.

Figure 3. Dot-plot gating strategy for acquisition and analysis. FSC vs SSC (A)
and FL3 vs FL4 (B).
9. Typical Data

9.1 Flow cytometry analysis

Flow cytometry analysis - data provided for demonstration purposes
only.

Figure 4. Flow analysis of exosomes. Cell culture exosomes, pre-enriched

using Total Exosome Isolation from PC3 Cell Culture Media (A) and human
plasma (B), were resuspended in PBS and bound to CD9-capture beads
during an overnight incubation. The following day the bead-bound
exosomes were indirect stained with primary antibody detection (CD9-
PE/CD81-PE).cytometry.
9.2 Performance Data
Limit of Detection (LOD), dynamic range and linearity of the kit was
assessed.
LOD is the lowest quantity of exosomes that is distinguished from the
absence of analyte (a blank value), and as reference, was
determined in >0,125 μg which corresponds with >1.5 x 108 vesicles.
The upper limit or saturation level was established in 8 μg. For both
technical specifications, exosome from PC3 cell culture media (12 x
108 vesicles / μl) was used (Figure 5).

Figure 5. Dynamic range of the assay analyzed by flow cytometry.

Relationship between background noise and specific signal at different
exosome concentrations.

Several measurements of multiple concentrations of lyophilized

exosomes were analyzed across the reportable range of the kit,
finding the linearity of the kit in a broad range of concentrations,
allowing fluorescence interpolation in the estimation of
concentrations.

9.3 Reproducibility

Intra assay:
Was determined calculating the deviation and the CV for each of
the samples by batch. Was analyzed the mean of all typical
deviations and CVs of 3 days for each lot. Finally, was obtained the
mean of the standard deviation and the CV of the three lots.

CV = 10%

Inter assay:
Was determined the mean of the 4 repetitions for each day and
compare them between each batch taking the standard deviation
and the CV. Was calculated the mean deviation thus obtained and
the CV of the three days.

CV = 11%
10. Notes

Technical Support
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