DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water

A simple assay is described using bisbenzimidazole (Hoechst 33258) to determine cellular DNA content in 96-well tissue cultures plates. At time points of interest, the plates are emptied of media and stored frozen. When the assay is to be performed, cultures are briefly incubated in distilled water and frozen again. This process lyses the cells and allows rapid and thorough mixing of the fluorochrome and cellular DNA. Freezing permits convenient storage of cultures until the time of assay. Experiments can be batched, further reducing processing time and giving better intra- and interexperimental standardization. The assay generates a linear standard curve for DNA fluorescence versus cell number, the range of which is appropriate for microculture wells. This enables the rapid and accurate measurement of cell number involving minimal processing time, making this assay well suited for cell proliferation studies.