Fluorescence microscopy of DAPI stained cells

Background 4'-6-Diamidino-2-phenylindole (DAPI) is known to form fluorescent complexes with natural double-stranded DNA, showing a fluorescence specificity for AT, AU and IC clusters. Because of this property DAPI is a useful tool in various cytochemical investigations. When DAPI binds to DNA, its fluorescence is strongly enhanced, what has been interpreted in terms of a highly energetic and intercalative type of interaction, but there is also evidence that DAPI binds to the minor groove, stabilized by hydrogen bonds beween DAPI and acceptor groups of AT, AU and IC base pairs.

Procedure Cells are grown in complete RPMI-1640. Harvested cells are washed once with PBS, and then resuspended in PBS containing 0.1 % Triton X (to induce holes in the cells' membrane = increase permeability) and incubated for 10 min on ice. Spin cells down and resuspend them at 5000 cells/µl in 4% PBS buffered paraformaldehyde solution containing 10 µg/ml 4'6-diamidino-2-phenylindole (DAPI, Sigma). 10 µl of this suspension are placed on a glasslide and covered with a coverslip. The morphology of the cells' nuclei is observed using a fluorescence microscope (Olympus BH Series) at excitation wavelength 350 nm. Nuclei are considered to have the normal phenotype when glowing bright and homogenously. Apoptotic nuclei can be identified by the condensed chromatin gathering at the periphery of the nuclear membrane or a total fragmented morphology of nuclear bodies. More than 150 cells are counted and the percentage of apoptotic nuclei determined.