Apoptosis can be detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, but not by TUNEL assay or sub-G0 DNA content.
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.


CONCLUSIONS
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.
Clarke
R G
RG
Department of Food Biophysics, Institute of Food Research, Norwich Research Park, Norwich, United Kingdom.
Lund
E K
EK
Johnson
I T
IT
Pinder
A C
AC
D000165:Acridine Orange; D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D002448:Cell Adhesion; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D019073:HT29 Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D007213:Indomethacin / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010718:Phosphatidylserines / Q000378:metabolism; D011419:Propidium; D011485:Protein Binding; D016192:Resting Phase, Cell Cycle
D017304:Annexin A5; D010718:Phosphatidylserines; D011419:Propidium; D004247:DNA; D000165:Acridine Orange; D007213:Indomethacin