Has the duration and temperature of lysis buffer been minimized for the comet assay?

The single cell‐gel electrophoresis also known as the comet assay, is a method of choice for measuring different types of DNA damage in individual eukaryotic cells. Performing the comet assay occupies three days, excluding the final image analysis. The comet assay includes: lysis overnight (day 1), electrophoresis and neutralization (day 2), staining (day 3) and then scoring using the fluorescent microscopy. Although the comet assay is a sensitive, specific and reliable methodology, yet, it is very time‐consuming. Therefore, the aim of this study was to minimize the duration of the comet assay by examining the effect of the lysis buffer temperature and the duration of the lysis step on the efficiency of this assay. Experiments were conducted using human dermal keratinocytes (HaCaT cells) as a model system, (untreated or treated with 50 μM H 2 O 2 ) to compare the lysis buffer temperature at 4 °C and room temperature for multiple time courses: 0.5 – 4 h and overnight. Treatments were performed in duplicates for each case. After the electrophoresis and neutralization steps were conducted, the percentage tail DNA on comets were scored using Comet IV software. A key finding was that there were no significant differences in the level of DNA damage between cell lysis at 4 °C for an overnight or for 30 min at room temperature. This finding suggests that it is possible to minimize the length of the comet assay by reducing the duration of lysis step to 30 min at room temperature. 1Effect of duration and temperature of lysis buffer on % tail DNA of HaCaT cells. Bars represent the mean ± SD of 300 individual cells for the different times (O/N, 4 h, 2 h, 1 h, or 30 min) at 4 °C vs. RT.