Re: Apoptosis model systems
Julie Auger (jauger@flowcity.bsd.uchicago.edu)
Sun, 05 Mar 1995 15:23:37 -0600
>Hello all,
>
>We are interested in beginning some work in the field of apoptosis and we
>want to try some of the methods described by Li and Darzynkiewicz.
>Our primary interest will be (human) keratinocytes, but we want to start
>with some positive control. Reading the instructions provided with the ONCOR
>kit lymphocytes treated with dexamethasone seems to be a model for inducing
>apoptosis. I would like to know if anyone is using this model, or which
>other model systems are used? Any advice would be appreciated.
>
>-----------------------------------------
>Piet EJ van Erp
>University Hospital Nijmegen
>dept. of Dermatology
>P.O. Box 9101
>6500 HB NIJMEGEN / The Netherlands
>E-mail: P.vanErp@derma.azn.nl
>Voice: +80-613548
>Fax: +80-541184
>-----------------------------------------
>
>
>
I have used dexamethasone as a positive control for apoptosis in mouse
thymocytes. I do not know if it works well on spleen or peripheral blood
lymphocytes. In this system, I have seen apoptosis within 4 hours of
culture. By 24hr almost all thymocytes are dead/apoptotic. The model
system that I have used to test all of our apoptotic assays is as follows:
1. Thymocytes kept at 4 C 24 hrs (neg contro - ususally less than 5%
deathl), 2. Thymocytes cultured in media 24 hrs (thymocytes will undergo
apoptosis to some degree is left in culture without any stimulation -
usually 20-30% death) , 3. Thymocytes cultured with 1uM dex 24 hrs (usually
80-90% death).
As most of our work is in immunology, this model system works well for us.
However, every cellular system seems to apoptose a little differently. I
have especially found differences in the kinetics of apoptosis. Some cells
die very quickly, some take longer. So how this model will apply to your
kerotinocyte system is unknown to me. I have also found that some apoptosis
detection methods described in the literature are not appropriate methods
due to the treatment of the cells to induce apoptosis. For instance, a
method that utilizes changes in membrane (HO342, 7AAD) cannot be used in
systems where cells are stimulated with PMA and ionophore.
Anyway, good luck and have fun.
Julie
********************************************************************************
Julie A. Auger voice: 312-702-9212 lab
Director, Flow Cytometry Facility 312-702-9261 office
University of Chicago fax: 312-702-3701
5841 S. Maryland Ave. MC-1089
Chicago, IL 60637 e-mail: jauger@flowcity.bsd.uchicago.edu
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