R G Clarke1, E K Lund, I T Johnson, A C Pinder. 1. Department of Food Biophysics, Institute of Food Research, Norwich Research Park, Norwich, United Kingdom.
Abstract
BACKGROUND: Induction of apoptosis in adherent cell lines is associated with cell loss from the substratum. In this study the adenocarcinoma cell line, HT29, treated with indomethacin (400microM) has been employed as a model system to demonstrate how flow cytometric analysis can be used to quantify the changes that occur during this process. METHODS: Adherent and floating cell populations have been analyzed independently for effects on cell number, cell cycle characteristics and apoptosis using TUNEL assay and Annexin V binding. In addition apoptosis has been assessed using DNA laddering and morphology. RESULTS: Apoptosis was detected in adherent cells treated with indomethacin using Annexin V binding but not by other techniques employed in this study. In contrast, analysis of "floating" cells revealed the presence of apoptotic cells both in control and indomethacin treated cells using all the techniques employed. However quantification by flow cytometry showed that a significantly higher proportion of control "floaters" were late apoptotic/necrotic rather than apoptotic. DISCUSSION: The data here illustrate the need to interpret measures of apoptosis in adherent cell lines with care and the value of using flow cytometric techniques in the quantitative evaluation of the process. Copyright 2000 Wiley-Liss, Inc.
BACKGROUND: Induction of apoptosis in adherent cell lines is associated with cell loss from the substratum. In this study the adenocarcinoma cell line, HT29, treated with indomethacin (400microM) has been employed as a model system to demonstrate how flow cytometric analysis can be used to quantify the changes that occur during this process. METHODS: Adherent and floating cell populations have been analyzed independently for effects on cell number, cell cycle characteristics and apoptosis using TUNEL assay and Annexin V binding. In addition apoptosis has been assessed using DNA laddering and morphology. RESULTS: Apoptosis was detected in adherent cells treated with indomethacin using Annexin V binding but not by other techniques employed in this study. In contrast, analysis of "floating" cells revealed the presence of apoptotic cells both in control and indomethacin treated cells using all the techniques employed. However quantification by flow cytometry showed that a significantly higher proportion of control "floaters" were late apoptotic/necrotic rather than apoptotic. DISCUSSION: The data here illustrate the need to interpret measures of apoptosis in adherent cell lines with care and the value of using flow cytometric techniques in the quantitative evaluation of the process. Copyright 2000 Wiley-Liss, Inc.
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