Cell Guidance Systems Exo-spin Mini-Columns Panduan Pengguna - Halaman 6

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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

Prepare 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.

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.

Carefully 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.

Centrifuge at 50 x g for 60 seconds. Discard the eluate.

Place the column into a 1.5 ml microcentrifuge collection tube. Apply 200 µl PBS

to the top of the column.

Centrifuge 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.

If any PBS remains above the top filter, repeat spin at the