Optimizing DNA staining by Hoechst 33342 for assessment of chromatin organization in living cells

In recent years, there has been an increasing interest for applications of fluorescence measurements to studies on many physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization. This type of approach involves both optimal conditions for DNA staining and the use of several investigation methods such as flow cytometry, image cytometry, laser scanning confocal microscopy and spectral imaging. In this context, this report describes a stoichiometric method for nuclear DNA specific staining, using bisbenzimidazole Hoechst 33342 associated with verapamil, a calcium membrane channel blocker. This method makes it possible to follow variations of nuclear DNA content in cells that are maintained alive.