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Re your FACStar+ 4-color FITC/PE/Tricolor/APC setup:
1. We also had a few initial problems with similar setups partly caused
by inaccurate assembly of the crossed mirrors at the FL3-1/FL3-2
junction. Having played with re-assembly of that over several hours,
I can tell you *don't* do it unless you really must. You should be
able to get simultaneous alignment of both sides just by adjusting the
iris positions on each side. Get the Tricolor side adjusted first.
2. We use B_D calibrite and Flow Cytometry Standards PE.Cy5 beads for
setting compensation in that kind of setup. Being able to see the
PE.Cy5 bead fluorescence will also give you a check on possible
failure of your tricolor staining. There is also a quirk to look out
for when setting the FL2/FL3-1 compensation; if the pulse widths are
not well matched, you can't accurately cancel out the spillover.
Watch the pulse display as well as the dot plot while doing the
setting. If the compensated pulse gets hollow in the middle with a
peak at either or both sides, adjust the iris opening.
3. I hope you have realized that the HeNe will excite the Cy5
component of the Tricolor and that that signal will come up in the APC
channel almost as brightly as it does in the Tricolor channel.
Hope some of that helps.
Frank.
\ / < The Flow Systems Laboratory
Frank Battye, \__/ <<<<< The Walter & Eliza Hall Institute
Dora Kaminaris,----------!!<<<<<<<< Victoria 3050, AUSTRALIA
Viki Lapatis and /!!\ <<<<< ph: 61_3_9345 2540 fax: 61_3_9347 0852
Jennie Parker o !! \ < in:: "facs_copy@wehi.edu.au"
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web