Nuclear Staining Dyes

Structure

DAPI (4',6-diamidino-2-phenylindole) (Figure 1) is a well-characterized blue-emitting fluorescent compound widely utilized for nuclear staining. DAPI permeates cell membranes and binds to the minor groove of A/T-rich dsDNA sequences, thus preferentially staining nuclei.

Fig. 1. Molecular structure of PureBlu DAPI Nuclear Staining Dye.

Excitation/Emission Spectra

When bound to dsDNA, PureBlu DAPI Dye has a maximum excitation wavelength in the ultraviolet range (359 nm), and the dye can be optimally detected in the blue channel with an emission maximum of 461 nm (Figure 2).

Fig. 2. DAPI excitation and emission spectra.

The characteristic Stokes shift between excitation and emission wavelengths is fairly wide for DAPI, making this dye an optimal choice when good spectral separation is desired to reduce fluorescence interference, for example, in chromatin counterstaining for immunofluorescence microscopy.

Fig. 3. Analysis of Jurkat and HeLa cells. A, flow cytometric analysis of Jurkat cell viability. Jurkat cells were heat shocked at 65°C for 5 min. Dead cells show positive staining with PureBlu DAPI. B, analysis of DNA content of Jurkat cells. Cultured cells were harvested, washed in phosphate buffered saline (PBS), and fixed in cold 70% ethanol for 2 hr at 4°C. The Jurkat cells were then washed in Bio-Rad Staining Buffer (BUF073) and stained with PureBlu DAPI Dye containing 0.1% octylphenol ethoxylate for 30 min. Cells were first gated to discriminate single cells and then plotted into histograms discriminating G1, S, and G2/M populations. All flow cytometry was carried out on a Bio-Rad ZE5 Cell Analyzer (12004279). C, HeLa cells, formaldehyde fixed and permeabilized, stained with Rat Anti-Alpha Tubulin Antibody (red) (MCA78G), Mouse Anti-Human CD49a Antibody, green (MCA1133), and PureBlu DAPI Dye (blue).

Specification

Alternative for Unfixed Samples

As an alternative to DAPI we also have PureBlu Hoechst 33342 Nuclear Staining Dye in a similar 'ready-to-reconstitute' format suitable for unfixed samples.

Binding

Hoechst 33342 is a well-characterized blue-emitting fluorescent compound widely utilized for nuclear staining. Hoechst 33342 permeates cell membranes and binds to the minor groove of A/T-rich dsDNA sequences, thus preferentially staining nuclei (Figure 4).

Fig. 4. Hoechst 33342 (in blue) binding dsDNA.

Excitation/Emission Spectra

When bound to dsDNA, Hoechst 33342 has a maximum excitation wavelength in the ultraviolet range (350 nm), and the dye can be optimally detected in the blue channel with an emission maximum of 461 nm (Figure 5). The characteristic wide Stokes shift between excitation and emission wavelengths for Hoechst 33342, make this dye an optimal choice when good spectral separation is desired to reduce fluorescence interference, for example, in chromatin counterstaining for immunofluorescence microscopy.

Fig. 5. Hoechst 33342 excitation and emission spectra. Maximal excitation at 350 nm (blue) and maximal emission at 461 nm (red).

PureBlu Hoechst 33342 Nuclear Staining Dye is compatible with fixed and unfixed cells. Compared to DAPI, it exhibits greater permeability toward intact cell membranes and is preferred when conducting experiments with unfixed live samples.

Fig. 6. PureBlu Hoechst 33342 use for cell cycle analysis on the ZE5 Cell Analyzer.

G0/G1 and G2/M peaks are separated by the S phase. Jurkat cells were fixed in cold 70% ethanol for 2 hours and then washed. The cells were then stained with Hoechst 33342 in buffer containing 0.1% octylphenol ethoxylate for 30 min prior to analysis. Samples were gated to remove doublets.

Specification

Preferred Choice for Unfixed Samples

Use PureBlu Hoechst 33342 Nuclear Staining Dye with fixed and unfixed cells. It has increased permeability toward intact cell membranes when compared to DAPI and therefore is preferred when using unfixed samples.