Chapter 11 DAPI Staining of Fixed Cells for High-Resolution Flow Cytometry of Nuclear DNA

This chapter discusses the use of 4', 6-diamidino-2-phenylindole (DAPI) for flow cytometry (FCM). DAPI has been used from many years. It proved to be a specific, highly fluorescent stain, very well suited for FCM of DNA in whole cells, in nuclei, and in chromosomes. The chapter presents the staining protocol that has been developed for use in cell suspensions after fixation with 70% ethanol. It is applicable in specimens that have to be stored before measurement. The procedure consists of a pretreatment of cells with citric acid plus Tween 20 to produce a suspension of isolated nuclei, followed by the addition of a solution of disodium phosphate or trisodium citrate with DAPI, which raises the pH and stains the DNA. This protocol is recommended for the accurate and reproducible measurement of the nuclear DNA, especially for the assessment of DNA content variations in cell populations affected by chemical or physical mutagens and for the discrimination of spermatogenic cells.