higher viability of stained cells. The additional ethyl group in certain Hoechst dyes (Hoechst 33342) renders them more cell-permeable. There are nuclei staining dyes that allow for viability of cells after staining. Hoechst 33342 and 33258 are quenched by bromodeoxyuridine (BrdU), which is commonly used to detect dividing cells. Hoechst 33342 exhibits a 10 fold greater cell-permeability than H 33258. Cells can integrate BrdU in newly synthesized DNA as a substitute for thymidine. When BrdU is integrated into DNA, it is supposed that the bromine deforms the minor groove so that Hoechst dyes cannot reach their optimal binding site. Binding of Hoechst dyes is even stronger to BrdU-substituted DNA; however, no fluorescence ensues. Hoechst dyes can be used with BrdU to monitor cell cycle progression. Hoechst dyes are commonly used to stain genomic DNA in the following applications: Fluorescence microscopy and immunohistochemistry, often with other fluorophores Flow cytometry to count or sort out cells. An example is the use of Hoechst dyes to analyse how many cells of a population are in which phase of the cell cycle Detecting DNA in the presence of RNA in agarose gels Automated DNA determination Chromosome sorting Hoechst efflux is also used to study hematopoietic and embryonic stem cells. As these cells are able to effectively efflux the dye, they can be detected via flow cytometry in what is termed the side population. This is done by passing the fluorescence emitted from the excited hoechst through both red and blue filters, and plotting hoechst red and blue against each other. Toxicity and safety Because Hoechst stains bind to DNA, they interfere with DNA replication during cell division. Consequently, they are potentially mutagenic and carcinogenic, so care should be used in their handling and disposal. Hoechst stain is used to sort sperm in livestock and humans. Its safety has been debated. See also References External links Spectral traces for fluorescent dyes Manual for Hoechst stains An online guide to fluorescent probes and commercial labeling technologies Staining dyes Fluorescent dyes DNA-binding substances