Cell Guidance Systems Exo-spin Mini-Columns User manual

User Guide
Exo-spin™ Mini-Columns
For rapid purification and clean up
of samples up to 100 µl.
Suitable for blood sera, cell culture
medium and labeled exosome samples
Cat EX03
Protocol Version 2.4

2
Contents
1. Storage 3
2. Product Components 3
3. Product Information 4
4. General Information 4
5. Protocol for using (EX03) Exo-spin™ columns 5
6. Troubleshooting 7
7. Purchase Notification 8

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Storage
Upon receipt, store at 4°C.
Correctly stored components are stable for at least 6 months following purchase.
Product Components
EX03-8
•8 x 1.0 ml Exo-spin™ columns and waste collection tubes
EX03-25
•24 x 1.0 ml Exo-spin™columns and waste collection tubes
EX03-50
•2 x 24 x 1.0 ml Exo-spin™ columns and waste collection tubes

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Product Information
Exo-spin™ columns may be used for isolation of intact exosomes from any sample
smaller than 100 µl. This includes blood sera, but not blood plasma. Exo-spin™
columns may be used for purification of samples which have been purified by other
methods, such as precipitation and ultracentrifugation, in cases where additional
cleanup is required to remove proteins and smaller molecules. Exo-spin™columns
are very effective for the clean-up of labeled exosome samples.
Exosomes isolated using Exo-spin™ columns may be used in a variety of
downstream applications including DNA and RNA studies as well as in functional in
vitro and in vivo exosome assays.
Plasma contains much higher protein levels than sera and can only be processed
directly using EX04 Exo-spin™ Midi columns. Also, a kit specifically for isolation of
exosomes from blood (EX02 Exo-spin™ blood) can be used for isolation of
exosomes from plasma.
General Information
Notes on Cell Culture
Fetal bovine serum (FBS) contains a large number of exosomes. Exosome-free FBS
should be used in cell culture experiments. Exosome-free FBS is available
commercially. Alternatively, we found that Vivaspin20 100,000 MWCO PES (GE
Healthcare) centrifugal concentrators or Millipore® UFC910024 Amicon® Ultra-15
Centrifugal Filter Concentrator with Ultracel® 100 Regenerated Cellulose
Membrane, NMWL: 100,000 can be used to efficiently remove exosomes from FBS,
which should be previously diluted 50% in PBS.
The number of exosomes that are obtained from a cell culture sample will vary
depending on a variety of factors. These include the cell line, the length of time the
medium is exposed to the cells, and the total number of cells in culture. Cancer cell
lines may produce higher numbers of exosomes than non-transformed cell lines.
Note on collection of samples
A review in 2013 by Witwer et al (Journal of Extracellular Vesicles (2013) 2: 20360)
indicates that the way samples are collected and handled prior to purification can
have a significant impact on the quality of purified exosomes.

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Protocol for purification of intact exosomes using Exo-spin™
columns (EX03)
Note
Exo-spin™ columns are supplied pre-equilibrated with mili-Q water containing 20%
ethanol. The column matrix should be re-equilibrated with PBS prior to use.
A maximum of 100 µl per column may be used.
A. Remove any cells and cellular debris
B. Column purify exosomes
Figure 1 Protocol overview

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Protocol
A. For primary samples, such as sera, which contain cells, a step is
required to remove cells and cell debris. This step may be omitted
for secondary samples being cleaned up which do not contain
cells.
1. Transfer 100 µl of starting sample to a centrifuge and spin at 300 × g for 10
minutes to remove cells.
A web-based tool for calculating centrifugal force (g) and Nomograph
are available on the Exo-spin™ product pages of www.cellgs.com.
2. Transfer supernatant to a new centrifuge tube and spin at 16,000 × g for 30
minutes to remove any remaining cell debris.
B. Purification of Exosomes
3Prepare the spin column prior to application of your sample
a. Remove the outlet plug and place the Exo-spin™ column into the waste
collection tube provided. Outlet plug must be removed before the
screw cap.
b. Using a micropipette, aspirate preservative buffer from the top of the
column and discard it. In order to prevent the column bed from drying,
proceed to the next step promptly.
c. Equilibrate the column by adding 200 µl of PBS and spin down at 50 x g for
10 seconds.*If any PBS remains above the top filter, repeat spin at the
same speed with 5 seconds increments. Do not spin at too high speed
or for too long as this may desiccate or compress the resin.
4Carefully apply the 100 µl exosome-containing sample to the top of the column
and place the column into the waste tube. Samples smaller than 100 µl should
be made up to 100 µl with PBS prior to applying the sample to the column.
5Centrifuge at 50 x g for 60 seconds. Discard the eluate.
6Place the column into a 1.5 ml microcentrifuge collection tube. Apply 200 µl PBS
to the top of the column.
7Centrifuge at 50 x g for 60 seconds. The 200 µl eluate contains the purified
exosomes.
*An example of a suitable centrifuge is the iFuge M08 from Neuation Technologies.

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Troubleshooting
My sample does not elute from the column.
Possible causes:
Ensure that the plug has been removed from the base of column.
If the column has been spun too fast, it will be compromised and subsequently not function
correctly. Use our online tool and Nomograph, available on the product pages, to calculate
the correct RPM for your centrifuge. Be aware that some centrifuges can’t provide the
required low speeds.
My sample contains a lower amount of exosomes than expected.
Possible causes:
Ensure that the columns do not dry out during the procedure. Any column that is spun for
too long or at too high speed may dry out. Spinning the column at too high speed may also
compress the resin used in the column. This may cause the column to work inefficiently. If
the column dries, reduce spin speed and/or time.
Make sure that the volumes indicated for addition of the sample to the column are adhered
to. The exosome containing fraction elutes in a peak as shown below. If the volume of
sample added to the column is too small, the exosomes will be retained within the column.
Adjust the volume of sample to 100 µl with PBS.
The yield of exosomes is dependent on a variety of factors, particularly the type of biological
fluid used as starting material. If media is used, the amount of exosomes present will vary
widely depending on the cell line and the length of exposure (conditioning) of the media.
My sample has no measurable exosomes.
Possible causes:
This is most likely caused by complete drying out of the column causing loss of functionality.
Ensure the columns are kept hydrated at all times.
Can I increase the elution volume?
This is not recommended as it will result in co-elution of ribonucleoprotein particles and
proteins.

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Purchaser Notification
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products as set forth in the Terms of Sale found on the Cell Guidance Systems
web site at www.cellgs.com/Pages/Terms_and_Conditions.html
If you have any questions, please contact Cell Guidance Systems.
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conveys to the purchaser the limited, nontransferable right to use the purchased
amount of the product only to perform internal research for the sole benefit of
the purchaser. No right to resell this product or any of its components is
conveyed. This product is for internal research purposes only and is not for use
in commercial services of any kind, including, without limitation, reporting the
results of purchaser’s activities for a fee or other form of consideration. For
information on obtaining additional rights, please contact [e-mail].
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