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