کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2596114 1562378 2011 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم محیط زیست بهداشت، سم شناسی و جهش زایی
پیش نمایش صفحه اول مقاله
High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay
چکیده انگلیسی

DNA interstrand crosslinks (ICL) are induced both by several cytotoxic anti-cancer drugs as well as by the chemical warfare agent sulphur mustard (SM). Although measurement of ICL formation could be used in risk assessment or provide valuable predictive information on the response of malignant cells to crosslinking chemotherapeutic agents, respectively, it is currently not applied due to lack of appropriate standardized methodology. Here we describe a fast and convenient procedure for detection of ICL in human peripheral blood mononuclear cells (PBMC) as high-throughput method, termed ‘reverse FADU assay’. This assay detects ICL based on the prevention of time-dependent alkaline unwinding of double-stranded DNA in a cell lysate that starts from single or double strand breaks. We have successfully established and optimized the reverse FADU assay by using human PBMC exposed to the model compounds mitomycin C, melphalan and SM. Our fully automated assay version is faster than currently used methods and possesses similar sensitivity. It operates in a 96-well format, thus allowing parallel analysis of multiple samples. Furthermore, we describe optimized protocols for sample preparation, with sample volume minimized to 100 μl of blood, storage and shipment conditions. We conclude that the reverse FADU assay is an attractive candidate method for monitoring DNA damage induced by DNA crosslinking agents.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Toxicology - Volume 280, Issues 1–2, 4 February 2011, Pages 53–60
نویسندگان
, , , , ,