[Cytometry] Acridine orange staining

Howard Shapiro hms at shapirolab.com
Wed Jul 1 21:04:16 EDT 2009

Zbigniew Darzynkiewicz wrote:
> If one wants most efficiently clean up the tubing from AO or other fluorochrome I suggest to run the sample containing 0.05M solution of caffeine in PBS. Caffeine has high affinity to form complexes with flat aromatic molecules such as AO or many other dyes and removes them from plastic surfaces or even from already stained cells very effectively by sequestering them in heterologous (caffeine:fluorochrome) complexes(See: Bedner et al. Caffeine dissociates complexes between DNA and intercalating dyes. Application for bleaching fluorochrome-stained cells for their subsequent re-staining and analysis by laser scanning cytometry. Cytometry 2001; 43:38-45.)[Nota bene: caffeine is very inexpensive - I guess! 
>   it is the by-product of de-caffeinization of coffee and tea]. 
Wait until you see the pricing on Jolt Sheath Fluid...On the other hand, 
those of us who work with intercalating dyes should be able to put 
prophylactic espresso makers on our grants.
> The myth that AO is irreversibly damaging instruments was developed over 25 years ago when AO was used to measure activation of lymphocytes. Then some reagent companies trying to convince the users to replace this very inexpensive AO test with the assays based on the monoclonal antibody (which costs ~$400) developed the scare tactics presenting AO as the instrument-damaging dye. For some time they even refused to continue warranty of the cytometers if the users were measuring cells stained with AO.
This is a very good point. Although some lymphocyte activation antigens 
are expressed within a few hours of stimulation of peripheral blood 
lymphocytes, increases in cellular RNA content can be detected as early 
as 12 hours after stimulation, using inexpensive dyes. There are now 
many alternatives to AO for DNA/RNA content measurement; Hoechst 33342 
and pyronin Y can be used to stain DNA and RNA in unfixed cells without 
compromising viability, and, during the past few years, a variety of 
visible-excited DNA-selective dyes and a few RNA-selective dyes have 
become available, providing considerable flexibility in terms of 
excitation and emission wavelengths. Sample prep is much easier than it 
would be if intracellular markers such as cytokines or Ki-67 or even 
surface antigens such as CD25, CD38, CD69, or CD71 were used as 
indicators of activation; fluorescence signals from RNA dyes are also 
substantially stronger than those from antibody-labeled activation antigens.

I've been threatening to do a comparative study of DNA/RNA dyes for 
ages. The opportunity has not arisen because I have done very little 
work on lymphocyte activation for the past few years, but my current 
interest in malaria has gotten me back in the DNA/RNA analysis business 
in a big way, since DNA and RNA content measurements alone can precisely 
identify the stage of development of malaria parasites. There are also 
enough new dyes out to make things interesting. Watch this space.


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