Apoptosis in HT29 cells

From: LAADAMS@am.pnu.com
Date: Fri Apr 25 1997 - 15:44:12 EST


Dear flowers,

HELP!!!  Has anyone out there been able to detect apoptosis in HT29 cells 
(or any other adherent tumor cell line) using TUNELing?  I have tried all 
the fixation/permeablization protocols I can find in the literature, and I 
have tried Boehringer Mannheim's In Situ Cell Death Kit and Phoenix Flow's 
Apo-BrdU kit.  (Thank you Phoenix for putting positive and negative 
controls in your kit!  At least I know the assay is working.)  I have 
verified the negative TUNELing by fluorescent microscopy in cells I know 
are undergoing apoptosis, i.e., I have seen PI positive/FITC negative 
apoptotic cells with my own eyes.  I can readily detect apoptosis in these 
cells by DNA laddering, Hoescht dye, and the appearance of a subdiploid 
peak with PI staining.  Since I would like to do other concomitent flow 
cytometric analyses along with the detection of apoptosis, I would rather 
not rely on something like laddering.

I have not tried Annexin since I am unsure of how to detach adherent cells 
without disturbing the plasma membrane, and I want to be able to determine 
apoptotic cells at all stages of apoptosis.  

ANY AND ALL SUGGESTIONS/IDEAS/COMMENTS ARE WELCOME AND APPRECIATED!  Thanks 
in advance!

Lisa Adams
Cancer Research
Pharmacia & Upjohn
Kalamazoo, MI
phone: 616-833-1556
FAX:   616-833-9763
  



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