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AO is a strongly fluorescing cationic dye which indeed
electrostatically attaches to the tubing and unless is thoroughly
washed out, interferes with analysis of the subsequent samples if the
latter have low fluorescence. There is nothing particular in this
respect, however, about AO. Other strongly fluorescing and cationic
dyes have similar, or even more pronounced (e.g. Rhodamine 123)
properties. We routinelly use AO in FACScan and in Cytofluorograph.
After each use of AO, prior to measurement of the immunofluorescently
labeled samples, we rinse the tubing by running the bleach solution
for 15 min, then PBS. High molarity salt solution (e.g. 2 M NaCl) is
even more effective in removal of the electrostatically attached
molecules than is PBS. When the sample tubing is very long (as it is
in some instruments), and the strong fluorochromes (charged
molecules) are frequently, and intermitently with immunofluorescent
samples, are used, it is advisable to have separate sample tubings
each dedicated to a particular dye, and replace them accordingly.
The stories about the miraculously potent deletorious effects of AO
spread by the manufactures of the reagents are most likely in support
of these reagents. There are several functional tests (e.g. mitogenic
stimulation of lymphocytes) which can be run with the use of AO at
minimal cost (the cost of reagents per test is less than 1 penny).
The same tests, however, run with monoclonal antibodies often cost
three orders of magnitude more.
Zbigniew Darzynkiewicz
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