Fluorometric determination of DNA in epidermis and cultured fibroblasts, using 4′-6-diamidino-2-phenylindole (DAPI)

Summary

4′-6-diamidino-phenylindole (DAPI) which is used for the detection of mycoplasmic infections of cell cultures or cytofluorometric investigations proved to be a sensitive agent for the fluorometric assay of alkali extracted DNA in cultured cells and human epidermis. Various experiments to optimize assay conditions led to the following procedure: 1 part of freeze dried tissue (1 mg/ml) and 1 part of 0.2 mol/l NaOH (w/v) were heated at 95° for 40 min. After cooling for 1 h to room temperature, 0.1 ml alkaline extract was mixed with 2.9 ml DAPI-solution (0.2 μg/ml in McIlvaine buffer containing 1 mmol/l MgCl₂). After 10 min in the dark the emitted fluorescence was estimated at 453 nm with an excitation at 360 nm. This assay procedure allowed a detection limit of 25 ng DNA.