Sirota Nikolai P, Zhanataev Aliy K, Kuznetsova Elena A, Khizhnyak Eugenii P, Anisina Elena A, Durnev Andrei D
Institute of Theoretical and Experimental Biophysics of Russian Academy of Sciences, Pushchino, Russia.
Zakusov Research Institute of Pharmacology of RAMS, Moscow, Russia.
Mutat Res Genet Toxicol Environ Mutagen. 2014 Aug;770:16-22. doi: 10.1016/j.mrgentox.2014.05.003. Epub 2014 May 27.
We performed an inter-laboratory study to determine the variation of comet assay results and to identify its possible reasons. An exchange of slides between Labs in different stages of the comet assay protocol was performed. Because identical slides, durations of alkali treatment and electrophoresis, and similar electric field strengths (2.0 V/cm and 2.14 V/cm) were used, we concluded that the observed inter-laboratory difference in the results is directly associated with the electrophoresis step. In Lab 1, mouse bone marrow cells were exposed to methyl methanesulfonate at concentrations of 10, 25 and 50 μM for 3 h at 37 °C. In Lab 2, cells the same as in Lab 1 were immobilized in LMA on slides and exposed to X-rays at doses of 3-8 Gy. We found that the transportation of slides after lysis or electrophoresis step, as well as different dyes used for scoring did not produce any significant effect on the results. No substantial difference in the data was also revealed when various software packages were used for image analysis. The temperature of the alkaline solution was shown to increase during electrophoresis and, besides, the temperature heterogeneity of the solution took place in the area of the platform, with a maximum in the middle of the chamber. The temperature heterogeneity could affect the rate of conversion of alkali labile sites into single stranded breaks. Thus, it was clearly indicated that real temperature variations during the alkali treatment and electrophoresis were an essential factor in the variability of the results between our Labs.
我们进行了一项实验室间研究，以确定彗星试验结果的差异并找出其可能原因。在彗星试验方案的不同阶段，不同实验室之间进行了载玻片交换。由于使用了相同的载玻片、碱处理和电泳时间以及相似的电场强度（2.0 V/cm和2.14 V/cm），我们得出结论，观察到的实验室间结果差异与电泳步骤直接相关。在实验室1中，将小鼠骨髓细胞在37°C下分别暴露于浓度为10、25和50μM的甲磺酸甲酯中3小时。在实验室2中，将与实验室1相同的细胞固定在载玻片上的低熔点琼脂糖中，并暴露于3-8 Gy的X射线下。我们发现，裂解或电泳步骤后载玻片的运输以及用于评分的不同染料对结果没有产生任何显著影响。当使用各种软件包进行图像分析时，数据也没有显示出实质性差异。结果表明，电泳过程中碱性溶液的温度会升高，此外，平台区域的溶液温度存在异质性，在腔室中间温度最高。温度异质性可能会影响碱不稳定位点转化为单链断裂的速率。因此，很明显，碱处理和电泳过程中的实际温度变化是我们各实验室之间结果变异性的一个重要因素。
