Re: Tunnel Assay

Neal Benson (
Thu, 17 Oct 96 10:22:10 edt

>I have a user who is trying an Assay called the Tunnel Assay. They are
>trying to detect Apoptotic cells that are of a specific phenotype.
>ie they need to be able to do surface marking and Tdt. The procedure
>they are using ends up stripping off their surface markers or at worst
>dimming them out.
>Any ideas?
>Jim Houston


Most procedures for TUNEL ("Tdt-Utp Nick End Labeling" - but I'm told that's
a misnomer anyway) are also designed to counterstain the DNA and have
ethanol as part of the fixation method, which can cause a loss of many
cell-surface antigens. Have them look at:

Sgonc R, Boeck G, Dietrich H, Gruber J, Recheis H, and Wick G: Simultaneous
determination of cell surface antigens and apoptosis. Trends in Genetics,
10:41-42, 1994.

The above uses a brief paraformaldehyde fixation followed by triton to
permeabilize and should work better to retain the surface. We have also had
success using the commercial product FACSLYSE from BD as a
fixation/peabilization reagent. See:

Syrjala MT, Tiirkainen M, Jansson SE, and Krusius T: Flow cytometric
analysis of terminal deoxynucleotidyl transferase. A simplified method.
American Journal of Clincial Pathology, 99:298-303, 1993.

Good luck,

Neal A. Benson
Biotechnology Program, University of Florida

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