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Of the remaining replies, a few (Tom Knapp, Aurora Biosciences; Tom Frey,
B-D Immunocytometry Systems) suggested using a different optical filter for
FL2 (PE) detection; is this a practical approach on a FACScan? Henry
Wortis (Tufts) suggested that a reduction in FL-2 and FL-3 amplification
might help, although Tom Frey (B-D) suggests that a change in PMT voltage
won't help, because PE signal (FL2) will only be detectable over PI (in
FL2) when the PE/PI ratio in FL2 is relatively high (i.e., fourth decade PE
staining, in his estimation). We can only attain this level of staining
with rare antibodies, so it is not likely to help in establishing a routine
procedure for FACScan use.
In summary, there doesn't appear to be a good resolution of this problem
other than to settle for inferior CVs with other DNA stains or to use a
multilaser instrument. I should mention that we have tried innumerable
permutations of fixatives, detergents, incubation times, and temperatures
without improving the CV of dyes like 7-AAD. If there are any other
suggestions from anyone who has successfully used such a method, I'd be
happy to hear them. Otherwise, back to the drawing board...
>>>>>>>>>>>>>>>> ORIGINAL MESSAGE:
Dear Flow Users: I am trying to develop a working method for two-color
surface staining plus DNA content analysis on a FACScan. In my hands, none
of the fixation/permeabilization methods that maintain surface
immunofluorescence generate adequate 7-AAD histograms (high CVs). I have
been concentrating on trying FITC/PE plus PI (ca. 0.5-1 microgram/ml). I
can compensate PE/PI at this level, and CVs are adequate, although
certainly not as good as PI at higher concentrations. The problem I appear
to be having is that (apparent) PE fluorescence disappears when PI is
added. I'm sure it's some compensation dilemma, but I can't seem to make
headway with this problem. Does anyone use such a method, and if so, can
you suggest a compensation protocol? I will repost a summary in the event
of multiple responses. Thanks for your help.
>>>>>>>>>>>>>>>> End of original.
Howard T. Petrie, Ph.D.
Assistant Member, Immunology Program
Memorial Sloan-Kettering Cancer Center
Box 341, 1275 York Avenue
New York NY 10021
phone (212)639-2149
fax (212)794-4019
e-mail: h-petrie@ski.mskcc.org
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