Measurement of cell numbers in microtiter culture plates using the fluorescent dye Hoechst 33258

A sensitive fluorimetric assay based on DNA binding of the fluorescent dye Hoechst 33258 was developed to quantitate cell numbers in microtiter plates. The cells were fixed and incubated with the dye solution. The bound dye was extracted with ethanol and fluorescence was measured in a spectrofluorimeter. The method is sensitive for as little as a thousand cells and is particularly useful for the normalization of enzyme-linked immunoassay (ELISA) data obtained from the same cells. Another major advantage of this new technique is that in contrast to previously described methods, it requires commonly available, inexpensive equipment.