Mutagenesis Advance Access originally published online on February 24, 2006
Mutagenesis 2006 21(2):105-114; doi:10.1093/mutage/gel004
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UVA-induced apoptosis studied by the new apo/necro-Comet-assay which distinguishes viable, apoptotic and necrotic cells
1Royal Cornwall Hospital NHS Trust, Sunrise Centre, 2Cornwall Dermatology Research Project, Knowledge Spa, Royal Cornwall Hospital, Treliske, Truro, Cornwall, UK and 3Institute for Molecular Biotechnology, Department of Single Cell and Molecule Techniques, Beutenbergstr. 11, D-07745 Jena, Germany
An adaptation of the Comet-assay was developed which enables the discrimination of viable, apoptotic and necrotic single cells by use of the common Annexin-V staining and a dye exclusion test on the cells already embedded in agarose gel on glass slides. Membrane integrity (Ethidium–Homodimer exclusion), cellular esterase activity (Calcein blue-AM) as well as translocation of phosphadidyl-serine (Annexin-V) were analysed using these stains. The advantage of the ‘apo/necro-Comet-assay’ is that the viability status of individual cells can be determined and correlated with the DNA fragmentation pattern (comet) formed by the same cells. Hence, DNA damage can be assessed and correlated with viable cells or cells undergoing early, mid- or late stage apoptosis or necrosis as identified by the staining pattern. The staining was verified using heat and etoposide-induced apoptosis. This technique, among others, was used to study whether apoptotic fragmentation interferes with repair kinetics measured with the comet assay following UVA exposure (doses up to 1280 kJ/m2) in the cultured human keratinocytes (HaCaT). Therefore, a time course of apoptotic events (phosphatidyl translocation and TUNEL fragmentation) was established and correlated to the DNA fragmentation in the comet-assay. Apoptotic cells were detected more than 8 h later. The combined three-colour staining method with the comet assay showed that there was no significant interference of DNA repair by apoptotic fragmentation processes since DNA repair was almost completed before the onset of apoptotic fragmentation. The apo/necro-Comet-assay reduces the general problem of false-positive results in genotoxicity tests using the Comet-assay.
* To whom correspondence should be addressed at: Royal Cornwall Hospital NHS Trust, Sunrise Centre, Royal Cornwall Hospital, Treliske, Truro, Cornwall, TR13LJ, UK. Tel: +01872 258342; Fax: +01872 252641; E-mail: nick.morley{at}rcht.cornwall.nhs.uk
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