Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain. IdisNa, Navarra Institute for Health Research.
Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain. IdisNa, Navarra Institute for Health Research.
Food Chem Toxicol. 2019 Oct;132:110671. doi: 10.1016/j.fct.2019.110671. Epub 2019 Jul 10.
The in vivo comet assay is usually performed in fresh tissues by processing cells immediately after collection, an approach that is not always possible from a logistical point of view. Although the comet assay has been applied to frozen rodent tissue samples on several occasions, there is currently no agreement on the best way to freeze and thaw them. We have tested two different thawing procedures and compared the levels of DNA strand breaks (SBs) and Fpg-sensitive sites in fresh and frozen (for up to year) liver, kidney and lung tissue samples, from untreated and methyl methanosulfonate treated rats. Tissues were snap frozen, stored at -80 °C and processed in such a way that the tissue remained frozen until the cells were in suspension. Our results showed that comparable levels of DNA SBs were detected in fresh and frozen liver and lung samples stored at -80 °C for up to 1 year and 3 months, respectively. In kidney, similar levels of SBs were detected either in fresh or in frozen tissues stored for up to 1 year. However, more studies are needed to control the variability observed in the Fpg-sensitive site levels in this tissue at the different freezing periods.
体内彗星试验通常在采集后立即处理新鲜组织中的细胞来进行，从后勤角度来看，这种方法并非总是可行。尽管彗星试验已经在多次冷冻啮齿动物组织样本上得到应用，但目前对于最佳的冷冻和解冻方法仍存在争议。我们已经测试了两种不同的解冻程序，并比较了新鲜和冷冻（最长可达一年）肝、肾和肺组织样本中 DNA 链断裂（SBs）和 Fpg 敏感位点的水平，这些样本来自未处理和甲基甲磺酸酯处理的大鼠。组织被快速冷冻，储存在-80°C 下，并以一种使组织在细胞悬浮之前一直保持冷冻状态的方式进行处理。我们的结果表明，在-80°C 下储存长达 1 年和 3 个月的新鲜和冷冻肝、肺样本中，均可检测到可比水平的 DNA SBs。在肾组织中，无论是新鲜组织还是冷冻组织，在储存长达 1 年的时间内，SBs 水平相似。然而，需要进一步研究来控制在不同冷冻期观察到的该组织中 Fpg 敏感位点水平的变异性。
