Hoechst Stain - Applications

Applications

A concentration of 0.1-12 µg/ml is commonly used to stain DNA in bacteria or eukaryotic cells. Cells are stained for 1-30 min at room temperature or 37 °C and then washed to remove unbound dye. A green fluorescence of unbound Hoechst dye may be observed on samples which were stained with too much dye or which were not washed thoroughly. Hoechst dyes are often used as substitutes for another nucleic acid stain called DAPI.

Key differences between Hoechst dyes and DAPI are:

• Hoechst dyes are less toxic than DAPI, which ensures a higher viability of stained cells
• The additional ethyl group of the Hoechst dyes renders them more cell-permeable.

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 together 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 together 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
• Detection of 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 known as 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.