Fluorometric determination of DNA in epidermis and cultured fibroblasts, using 4'-6-diamidino-2-phenylindole (DAPI).
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 degrees 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 microgram/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.
D002478:Cells, Cultured; D003080:Cold Temperature; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004817:Epidermis / Q000032:analysis*; D004996:Ethidium; D005347:Fibroblasts / Q000032:analysis*; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D004247:DNA; D004996:Ethidium