Fluorescent dyes for cell viability: an application on prefixed conditions

In recent years increasing attention has been given to apoptosis for its ro le in pathologic, organogenetic and homeostatic phenomena. Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyse cell culture viability. In fact, they resp ectively show specificity for living, apoptotic and late apoptosis/necrosis states. We explored whether HO, AO and PI can be used on prefixed monolaye rs of three commonly used cell lines. Here we mainly describe the metachrom atic effects obtained by fluorescence microscopy with double and triple dye combinations. Furthermore, we propose an easy staining method in which a b alanced sequential treatment with HO, AO and PI allows identification of di fferent viability states onto fixed cells by using a long-pass FITC filter. This method extends the spectrum of suitable applications for these dyes i n fluorescence viability detection onto previously fixed (prefixed) samples .