RE: Accutase for detachment for cell monolayers

For those interested in detachment of monolayer cells,

The phenomenon Kevin describes is likely to be caused by the formation of
inter-cellular junctional contacts between cells and contacts between cells
and the culture flask. This formation is time and cell line dependent. In
general: the longer you culture, the more junctions are formed, the more
effort it takes to detach the cells. Several protein families of cell
adhesion molecules (CAMs) play an important role in the formation of these
contacts, amongst are cadherins. Cadherins are essential for the maintenance
of the cellular morphology of epithelial cells. The homophilic mediate
adhesion of these molecules is Ca2+ dependent. This explains why EDTA or
EGTA facilitate epithelial cell detachment. The addition of EDTA to accutase
is very likely based on that principle rather than an increased activity.
Although I have now no knowledge about the composition of accutase or
accumax, chelating Ca2+ can reduce enzyme potentials, since Ca2+ can protect
proteolytic enzymes from auto degradation (e.g. trypsin). 

For those still working with trypsin/EDTA, it is important to wash the cells
before detachment. We harvest the old culture medium and spin cell debris
down (1000g). The supernatant can be used to block trypsin activity. Many
epithelial cancer cell types (breast, ovary, colon) produce tumour
associated trypsin inhibitors (TATI, see PubMed), which is released into the
medium. Cells are washed by Hank's Balanced Salt Solution, without Ca2+,
just by placing the culture flask in incubator at 37C. After 5 - 10 min.
cells already start to "round-up". This can be clearly observed using a
phase-contrast microscope. Withdraw the HBSS and add trypsin/EDTA
(0.025%/0.5 mM). Most cell lines detach within several minutes. Add the "old
medium", harvest and wash.

Several years ago we did a small study about the affect of trypsin on the
detection of cell surface molecules (or at least their epitopes recognized
by monoclonal antibodies) expressed by ovarian cancer cell lines. Trypsin
showed to be less harmful than expected.

Limited loss of nine tumor-associated surface antigenic determinants after
tryptic cell dissociation.
Cytometry. 1995 Mar 1;19(3):267-72. 

Best regards,

Willem Corver

--------------------------------
Willem E. Corver, BSc, PhD
Researcher
Department of Pathology
Leiden University Medical Centre
P.O. Box 9600, Building 1, L1-Q
2300 RC  Leiden
The Netherlands
Tel.: +31 71 5266604
Fax.: +31 71 5248158
--------------------------------

----Original Message-----
From: C. Kevin Becker [mailto:ckb@innovativecelltech.com]
Sent: woensdag 10 november 2004 22:52
To: cyto-inbox
Subject: Re: Accutase for detachment for cell monolayers


Dear Giovanna,

Detaching adherent cells is not an exact science because we are dealing with
a biological system that is not always predictable because of unknown
variables.  We at ICT have discovered for instance that certain cell lines
when they are at a low passage number will lift off immediately with
Accutase, but at a high passage number are difficult to lift off and more
Accutase is required.

In addition, Accutase will not lift off all known cell lines as trypsin will
not.  I would recommend you try our other product for cell detachment and
tissue dissociation, Accumax, which has a higher concentration of enzymes in
it.  If this does not work another interesting phenomena we have just
discovered, is if enough EDTA is added to the Accumax (which contains none)
to give a .5mM concentration, the activity of the enzymes can be potentiated
by up to a factor of 3.

Please let me know if we can be of any more assistance.

C. Kevin Becker
Innovative Cell Technologies, Inc.
6790 Top Gun St. Suite 1
San Diego, CA 92121 USA
858 587-1716
Fax 858 453-2117
www.innovativecelltech.com
No pigs in our products!-Accutase/Accumax


----- Original Message -----
From: "Giovanna Farruggia" <giovanna.farruggia@unibo.it>
To: cyto-inbox
Sent: Monday, November 08, 2004 8:20 AM
Subject: accutase for detachment for cell monolayers


> Dear Flo,
> we are trying to use accutase to detach caco cells from very confluent
> monolayers to perform immunofluorescence assays in flow cytometry, and we
> have a lot of problem, lot of cluster and debris and it was very difficult
> to find scatter signals. Does anybody has experience with this product and
> some suggestions? Thank you a lot
>
> Giovanna Farruggia
> Dip. Biochimica "G. Moruzzi"
> Universita' di Bologna
> Via S. Donato 19/2
> 40127 Bologna
> tel. 051 2095626
> fax 051 2095627
>
>