The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Letter
Evaluation of effect during cell isolation process in alkaline comet assay using epidermal skin cells
Tomoyasu ToyoizumiMika WatanabeHajime SuiYuzuki NakagawaRyo OhtaKohji Yamakage
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2012 Volume 37 Issue 6 Pages 1267-1273

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Abstract

The aim of the present study was to evaluate the effect of the cell isolation process in the alkaline comet assay using epidermal skin cells. When we explored the cell isolation method for the alkaline comet assay using the 3-dimensional (3D) human epidermal skin model, we found that DNA damage and cytotoxicity were induced during the cell isolation process. In particular, trypsin 5 min treatment with ethylene diamine tetraacetic acid (EDTA) showed about 5 times %DNA in the tail value compared to without EDTA treatment. In general, EDTA is commonly used for cell isolation, but it is known to induce genotoxicity due to secondary effects. We therefore evaluated the effect of EDTA and pH in the alkaline comet assay on a monolayer culture of rat keratinocytes. As a result, there was a significant increase of %DNA in tail values by treatment with 0.1 w/v% EDTA for 60 min; however, there was no difference in the %DNA in tail values between 0.1 w/v% EDTA/PBS(-) (pH 6.8) and 0.1 w/v% EDTA/PBS(-) (pH 7.4). These data imply that there is a need to control the EDTA conditions for cell isolation in the epidermal skin cells.

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© 2012 The Japanese Society of Toxicology
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