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When is it best to use Hoechst vs. DAPI for nuclear staining? They seem to be very similar on paper. Are there situations where one is clearly preferable?

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3 Answers 3

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The Hoechst 33342 dye is similar to DAPI in that both are UV-excited, minor groove-binding, and emit signals proportional to total DNA content. Both are maximally excited around 355 nm and emit around 460 nm. A UV light source is required, which may harm the cell. However, this is only a risk with Hoechst, as DAPI requires the cells to be fixed and/or permeabilized, which is incompatible with life. DAPI is more stable, but Hoechst is brighter. Both are subject to photo-bleaching after long exposure.

There is another newer DNA dye called DRAQ5 that makes up for all of the issues I mentioned earlier - it is bright, quite photo-stable, can be used in live cell imaging, and its excitation and emission wavelengths are in the far red end of the spectrum, meaning that no UV is needed. I found a good comparison chart at http://www.biostatus.com/product/draq5/live_cell_comparison_chart/ if you're interested. I have no connection to them, by the way, I've just used DRAQ5 a lot in the past.

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Sorry, but this answer is wrong: Most microscopes have a dedicated DAPI channel and both DAPI and Hoechst can very well be used together with FITC-like dyes. Also, you are missing the main point here: Hoechst can stain live cells, while DAPI can't. This is the main reason why people choose one over another. -1 not for that, but for the advertisement. I don't think this belongs here. –  Eekhoorn Nov 26 '12 at 9:41
    
-1: DAPI is absolutely compatible with GFP, FITC and Alexa 488.\ –  nico Nov 26 '12 at 18:57
    
my bad, edited to reflect reality. –  MattDMo Nov 26 '12 at 22:20
    
Use dapi for fixed cells, it's very bright, very specific, will let you know if you have mycoplasma contamination in your culture, has a good shelf life and is generally pretty inexpensive. If you have to do live cell imaging use hoechst and like @user1850479 stated there are multiple types, they are all generally less toxic. If you can't use a blue nuclear staine Draq works good, it's a little dim I've found but it is in far red range, it's relatively non/toxic, does not have as good a shelf life as the other I mentioned here, also it's really expensive $$$$$$$!!!! –  rhill45 Nov 9 at 3:46

I realize this is an older topic, but I'd like to clarify some misconceptions:

1) DAPI is usually referred to as a semi membrane-permanent dye because its penetration through viable cell membranes is concentration dependant. At lower concentrations it is mostly excluded from viable cells, while at higher concentrations it stains live and dead cells about equally.

2) Hoechst comes in both membrane permanent and impermanent varieties. Make sure you order the correct one for your application!

3) It is not necessarily true that Hoechst is preferred for viable cells. Hoechst has very slow diffusion through thick tissue for instance, so for in vivo imaging applications (or in tissue sections), DAPI often stains viable cells much better than the (much heavier) 33342 because it is better able to diffuse through tissue. For isolated cells however, Hoechst 33342 can much more rapidly stain viable nuclei (seconds vs. minutes for DAPI).

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DAPI and Hoechst 33342 (there are different Hoechst dyes, 33342 is one of the most commonly used) have very similar spectral characteristics. The only point is that DAPI is much better excited at 405 nm than Hoechst. Some microscopes and flow cytometers nowadays have 405 nm lasers or LEDs instead of UV sources, so this can become relevant.

The main difference and the main reason to choose either is that Hoechst can be used to stain living cells as it can pass the cell membrane, whereas DAPI can't. DAPI requires fixed and permeabilized cells.

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Does this imply that Hoechst stains are more hazardous to the researcher or the environment? If DAPI can't enter living cells but Hoechst can, are there concerns about mutagenicity (similar to e.g. ethidium bromide)? –  Tim Smith May 4 '13 at 18:18
    
Yes, indeed. But we treat Hoechst and DAPI the same way in our lab. –  Eekhoorn May 5 '13 at 9:11

protected by Chris Nov 7 at 23:21

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