DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe that forms a fluorescent complex by attaching in the minor groove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. DAPI is commonly used as a nuclear counterstain in fluorescence microscopy, flow cytometry, and chromosome staining.

DAPI's spectral properties make it ideal for use with green, red, and far-red fluorophores in multicolor experiments. It is less membrane-permeant than Hoechst dyes and is typically used to stain fixed cells. DAPI is also often used as a component of a mounting medium, for example, our LumiMount DAPI, to counterstain cell nuclei before imaging.

Here, we describe our experience of Lumiprobe's DAPI dye use for staining Madin-Darby canine kidney (MDCK) cells.

Protocol

1.      MDCK cells were seeded on sterile glass coverslips in a plate/culture dish and cultured in DMEM/F12 (1:1) supplemented with 5% fetal bovine serum and 1% glutamine at 37 °C in 5% CO2 overnight.

2.      After cells reached ~70-80% confluency, the media was removed, and the cells were washed twice with pre-warmed PBS.

3.      Cells were fixed with warm 4% formaldehyde solution for 15 minutes, followed by three washes with PBS for 5 minutes each.

4.      Cells were stained with a DAPI solution in PBS (at a DAPI concentration of 35-600 nM) for 5 or 30 minutes at 37 °C in the dark, followed by three washes with PBS for 5 minutes each.

5.      Cells were visualized using an epifluorescence microscope (Zeiss Axiovert, 20× LD-A-Plan) or a confocal microscope (Zeiss LSM900, 63×/1.25 Plan-Neofluar), with excitation at 405 nm and an emission detection range of 412-605 nm.

Results of staining

We varied the incubation time (5 and 30 minutes), the DAPI concentration (35-600 nM), and the microscope settings to determine the optimal conditions for imaging MDCK cell nuclei.

Figure 1. Testing the time of cell incubation with 300 nM DAPI solution.

Cells were imaged using the same imaging settings, which were optimized for a 30-minute incubation. The scale bar is 50 μm.

Figure 2. Selection of DAPI concentrations for staining cells for confocal microscopy.

Confocal microscope images of cells stained with different DAPI concentrations and imaged with the same imaging settings (optimal to 300 nM concentration). The scale bar is 50 μm.

Figure 3. Selection of DAPI concentrations for staining cells for confocal microscopy.

Confocal microscope images of cells stained with different DAPI concentrations and imaged at optimal imaging settings for each concentration of DAPI. The scale bar is 50 μm.

Figure 4. Selection of DAPI concentrations for staining cells for epifluorescence microscopy.

Fluorescence microscope images of cells stained with different DAPI concentrations and imaged with the same imaging settings (optimal to 300 nM concentration). The scale bar is 200 μm.

Conclusion

We found that incubation for 30 minutes yields a more intense and uniform signal, as well as a more favorable nucleus/cytoplasm signal ratio. For confocal microscopy, which allows the laser power and signal amplification to be varied over a wide range, concentrations as low as 35 nM are acceptable. For epifluorescence microscopy, the optimal concentration range is from 150 to 600 nM.