Use of Acridine Orange in killing assay. ngg1 at
Wed Nov 20 05:04:13 EST 1996

At 09:30 18/11/96 PST, Raffi Manoukian wrote:
>Hello Flowers,
>     This is my first time using "the list" and I think it's great, 
>especially since I am a newcomer  to the world of flow cytometry. I'm trying 
>to adapt an old microscopic technique that uses acridine orange in a 
>neutrophil killing assay, where candida or staph. a. is used as the target. 
>A.O. is supposed to passively enter the effector cells, and shift from green 
>to red fluorescence when phagocytosed targets are killed. Can anyone out 
>there explain this to me in a little more detail and tell me if it would 
>work with flow cytometry?
>Raffi Manoukian
>Royal Victoria Hospital
>Montreal, Quebec, Canada

Hello Raffi,

I have been working for a while with acridine orange to assess phagocytosis
of bacterial cells by rainbow trout macrophages.
The principle of shift in colour (Strugger's effect)is relatively simple: AO
binds to single stranded nucleic acids as a dimer, which fluoresces red
colour, and to double stranded nucleic acids as a monomer, which fluoresces
green. This has to do with the accesibility of AO molecules to the binding
Therefore, if a bacterial or yeast cell is viable, AO monomers bind to DNA
and as a result green fluorescence mainly is produced. However, if cells are
damaged, the binding sites in the DNA will be more exposed and AO will bind
in dimers, with red fluorescence as a result.
For this purpose you need to stain the cell under (supra)vital conditions
(similar pH, low AO concentration) not to disturb the DNA during the
staining process. However, if you stain under these conditions, you'll see
that in the cytoplasm there are other red structures, the lysosomes and/or
phagolysosomes, which accumulate AO in high concentrations-dimers. This is a
real problem in microscopy. I don't know whether flow cytometry would manage
to differenciate between these red vacuoles and red, non-viable,
bacterial/yeast cells.

Another technique to assess  killing is by FITC labelling of yeast/bacterial
cells and then challenging the PMNs. After reaction time, lyse PMN's and
stain with propidium iodine. Viable cells will exclude PI and fluoresce
green and non-viable cells will take up PI, staining red. I have no
experience, though, with this technique but there might be several
references with this technique (check for the proccedings book of the "2nd
international congress on phagocytes", Pavia, Italy, 4-7th September 1996,
where a good presentation of this technique was given).


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