Trypsinization Procedure for Corning® Transwell® Inserts CLS-AN ...

Trypsinization Procedure for Corning ® Transwell ® Inserts
Supplies
1. Cell culture medium. This should contain all of the additives (sera,
glutamine, etc.) required by the chosen cell line.
2. Calcium- and Magnesium-Free Phosphate-Buffered Saline CMF-PBS
(10mL). This simple salt solution is used to maintain proper pH and
osmotic balance while the cells are being washed to remove protease
inhibitors that are found in FBS.
3. 0.05% Trypsin/EDTA (0.05%) solution: Trypsin is normally used in
concentrations ranging from 0.05% to 0.25%. Working concentrations are
usually determined by using the lowest trypsin concentration that can
remove the cells from the substrate and give a single cell suspension in a
relatively short time (5 to 10 minutes). Trypsin solutions are often
supplemented with other enzymes (collagenase) or chelating agents
(EDTA) to improve its performance.
4. 15mL disposable screw cap centrifuge tubes (Corning Catalog # 430055 or
430788)
5. Hemacytometer
6. Centrifuge
7. Phase contrast microscope
8. 1, 5, 10 and 25mL pipettes - Corning Catalog # 4485, 4487, 4488 and 4489

Recommended buffer
and trypsin volumes
6.5mm Transwell insert:
add 0.1ml to the top, 0.6ml
to the well bottom;
12mm Transwell insert:
add 0.3ml to the top, 1.5ml
to the well bottom;
12mm Snapwell insert:
add 0.3ml to the top, 3.5ml
to the well bottom;
24.5mm Transwell insert:
1 add 1.0 ml to the top,
1.5ml to the well bottom.
Procedure
1. Aseptically aspirate spent media from the bottom well of the plate and from
inside the Transwell ® insert.
2. Rinse the top and bottom of the Transwell insert 2 to 3 times with PBS to
remove any serum (contains trypsin inhibitors). Review chart on left for
recommended buffer volumes.
3. Add Trypsin/EDTA to the bottom of the well and Transwell insert. Use the
same liquid volumes as above. Incubate for 10 minutes in a 37°C
incubator, containing 5% CO 2 .
4. After 5 minutes, view the cells under a phase contrast microscope for
detachment. Generally, the lower the trypsin concentration and the less time
the cells stay in trypsin the better. Once the cells look rounded or detached
(approximately 10 minutes), remove the Trypsin/EDTA containing cell
suspension and add to a 15mL centrifuge tube containing 5mL of serumcontaining
culture medium to inactivate the trypsin. (For serum-free culture
use a trypsin inhibitor.)
5. With the cover on the plate of the Transwell inserts, sharply hit the side of
the plate with your hand to detach cells from the membrane. Rinse the top
and bottom of the Transwell insert with Trypsin/EDTA to remove any
remaining cells. Add the cell suspension to the centrifuge tube.
6. Observe Transwell inserts under a phase contrast microscope to ensure that
all of your cells have been removed. Perform a second trypsinization if
there are large numbers of cells still remaining on the membrane by
repeating the steps 3 to 5.
7. Gently vortex the cells to resuspend and then remove an aliquot of cell
suspension and count with a clean hemacytometer. Centrifuge cells for 5 to
10 minutes at 100xg. Remove media from the centrifuge tube, leaving the
cell pellet, and resuspend cells in the appropriate amount of growth media.
For additional product or technical information, please visit our web site at
www.corning.com/lifesciences or call 1-800-492-1110. International
customers can call 978-635-2200.
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Corning and Transwell are registered trademarks of Corning Incorporated, Corning New York
Snapwell is a trademark of Corning Incorporated, Corning New York 4/03