Hoechst stain

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.

Chemical structure of Hoechst dyes
Excitation–emission spectra of Hoechst dyes
Hoechst 33258 (magenta) bound to the minor groove of DNA (green and blue). From PDB : 264D ​.
Transmission image of HeLa cells, with overlay of Hoechst 33258 staining (blue). The leftmost cell is in the prometaphase stage of mitosis ; its chromosomes fluoresce brightly because they contain highly compacted DNA.
Fluorescent image of cultivated neutrophils isolated from venous blood of human with Alzheimer Disease. Sample was treated with Hoechst 33342 dye that is used to stain DNA. The picture shows the release of DNA by a neutrophil as foggy area in the center of the view field indicating the spontaneous activation of neutrophil extracellular traps formation in AD patients that is not usually observed in healthy mates.