Re: simultaneous surface/DNA analysis
T. Vincent Shankey (tshanke@bsd.meddean.luc.edu)
Wed, 26 Apr 1995 18:39:58 -0500 (CDT)
We are measuring two intracellular antigens plus DNA on a Coulter XL with
one laser, using cytokeratin-PE, nuclear antigen-FITC and DNA with PI.
Aside from some compensation problems (log versus linear signals), this
set-up is working well (like Jim Jacobberger, I could provide an
extensive discourse here on problems and pitfalls of fixation,
permeabilization and staining of intracellular antigens, but these are
not relevant to your question). This would suggest one possibility that
you could try, as you indicated, doing FITC, PE and PI (for DNA) on one
laser, and Texas Red on the other laser. The problem with the strong PI
stain can be reduced by using a 630 or 640 long pass (6 cavity) filter in
front of the PI photomultiplier (an idea of Ken Bauer's) - this separates
the PI signal further from the PE, and with PI/DNA there's plenty of
signal left 20 nm plus above the fluorescence peak. Another possibility
is to use UV for one antigen (AMCA) plus DNA (PI- sometimes it is
forgotten that PI bound to DNA absorbs very nicely in the UV and emits at
610 nm). I hope this has been useful.
Vince Shankey
Loyola Univ. Med. Cntr.
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