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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web