Apoptotic cells are strongly labeled in TUNEL assay only when they have a large number of DNA strand breaks. This is the case when DNA fragmentation is very extensive, which occurs when internucleosomal DNA sections are cleaved. However, in some cell types (often of epithelial or fibroblast lineage) or instances DNA fragmentation stops at the initial step i.e. generating 300 - 50 kb DNA sections, and does not progress into intenucleosomal (~180 bp) sections. In the TUNEL assay such cells are only weakly labeled. It should be noted that with most TUNEL kits (including APO-BRDU) a positive control cells are provided. They are camptothecin-treated leukemic cells which are expected to have internucleosomal DNA cleavage in about 30-40 % of the cell population (S phase). This control, if found to be TUNEL positive, provides an assurance that the kit is OK. Furthermore, in the case when the control is positive but the investigated apoptotic cells are negative, it provides evidence that DNA fragmentation in the studied cells did not progress into internucleosomal DNA sections. Zbigniew Darzynkiewicz, M.D., Ph.D. Director, Brander Cancer Research Institute New York Medical College 19 Bradhurst Ave. Hawthorne, NY 10532 tel: 914-347-2801 fax: 914-347-2804 http://www.geocities.com/z_darzynkiewicz
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