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.

DAPI's blue emission is convenient for microscopists who wish to use multiple fluorescent stains in a single sample. There is fluorescence overlap between DAPI and green-fluorescent molecules like fluorescein and green fluorescent protein (GFP), or red-fluorescent stains like Texas Red, but using spectral unmixing or taking images sequentially can get around this.

Apart from labelling cell nuclei, the most popular application of DAPI is in detection of mycoplasma or virus DNA in cell cultures.


DAPI (magenta) bound to the minor groove of DNA (green and blue)




Stock Solutions

DAPI Stock Solution (5mg/ml or 14.3 mM):

DAPI 10 mg
Dimethylformamide (DMF) 2 ml
Mix to dissolve and it may take sometime to completely dissolve.
Aliquot and store in –20C.

DAPI Working Solution (100ng/ml or 300 nM in PBS):

DAPI stock solution 2 µl
PBS 100 ml

Store this solution at 4C in brown bottle or wrapped with aluminum foil to protect from light (to achieve stronger staining, use 4 µl stock solution or reduce PBS amount to 50 ml).

Procedure

Cells are grown as usual. 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 different time on ice. The staining time depends on the type of sample and varies from 5-30 min. After incubation spin cells down and resuspend them at 5000 cells/µl in 4% PBS buffered paraformaldehyde solution containing 100ng/ml 4'6-diamidino-2-phenylindole (DAPI). 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 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.