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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