Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique

Abstract

The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4′,6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.