Hoechst 33258 is widely used in biological and medical research as a fluorescent stain for DNA. It binds specifically to the minor groove of double-stranded DNA, particularly regions rich in adenine and thymine. This property makes it a valuable tool for visualizing chromosomes and nuclei in fluorescence microscopy, aiding in the study of cell division, nuclear morphology, and DNA content analysis. It is also employed in techniques like flow cytometry to sort and analyze cells based on their DNA content. Additionally, Hoechst 33258 is used in assays to detect apoptosis, as it can distinguish between live and dead cells based on changes in nuclear staining patterns. Its ability to penetrate cell membranes and stain DNA without requiring cell fixation makes it a convenient choice for live-cell imaging studies.