TUNEL ASSAY In culture, at least, not all types of cell proceed to the final stages of apoptosis. Although their nuclear morphology is that of apoptotic cells, they do not degrade their DNA to produce a DNA ladder (Oberhammer F, Wilson JW, Dive C, Morris ID, Hickman JA, Wakeling AE, Walker PR, Sikorska M. Apoptotic death in epithelial cells, cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J.1993; 12: 3679-3684. Ormerod MG, O'Neill CF, Robertson D, Harrap KR. Cisplatin induces apoptosis in a human ovarian carcinoma cell line without concomitant internucleosomal degradation of DNA. Exp. Cell Res. 1994; 206: 231-237). The TUNEL assay will detect the strand breaks produced during the initial stages of DNA degradation to 30-50 kbp. (as opposed to oligomers of 200 bp). The number of strand breaks, and hence the labelling, will be far lower than that observed after more extensive DNA degradation (Cain K, Inayat-Hussain SH, Wolfe JT, Cohen GM. DNA fragmentation into 200-250 and/or 30-50 kilobase pair fragments in rat liver nuclei is stimulated by Mg2+ alone and Ca2+/Mg2+ but not by Ca2+ alone. FEBS Lett. 1994; 349: 385-91. Chapman RS, Chresta CM, Herberg AA, Beere HM, Heer S, Whetton AD, Hickman JA, Dive C. Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells. Cytometry 1995; 20: 245 56). I would recommend that you look at the molecular weight of the DNA by conventional gel electrophoresis (to check for the DNA ladder) and by either pulse field gel or field inversion gel electrophoresis (PAGE or FIGE). In our study of some cultured ovarian cells, we did not observe a DNA ladder but we did find that the DNA had a MW of about 30 kbp. Be sure that you include both the attached and detached cells from the culture. If the apoptotic cells are not producing a DNA ladder, I would explore the use of the Annexin V assay to quantify the apoptotic cells. Michael Ormerod 34 Wray Park Road Reigate RH2 ODE Telephone: voice & FAX: (0)1737 241726 Web site: http://ourworld.compuserve.com/homepages/Michael_Ormerod
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