There is a considerable Stokes shift between the excitation and emission spectra that makes Hoechst dyes useful in experiments in which multiple fluorophores are used.
[3] The dyes bind to the minor groove of double-stranded DNA with a preference for sequences rich in adenine and thymine.
Although the dyes can bind to all nucleic acids, AT-rich double-stranded DNA strands enhance fluorescence considerably.
Thus, these stains are often called supravital, meaning that live cells survive a treatment with these compounds.
[citation needed] A concentration of 0.1–12 μg/ml is commonly used to stain DNA in bacteria or eukaryote cells.
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.
As these cells are able to effectively efflux the dye, they can be detected via flow cytometry in what is termed the side population.