I've seen the same thing on my FACSCalibur. The problem is that the instrument
is compensating FL3/FL1 behind your back...without your knowledge... or
approval. The nerve! I can't tell you how it is done exactly, but FL1 is
compensated out of FL3 based on the amount of compensation of FL2-FL1 and
FL3-FL2. I would much rather be able to do this on my own, but since FL1 signal
shouldn't (nudge, wink) spill into FL3, we are not given that capability with
the Calibur. There's not really much you can do about it, except maybe run at
higher voltages on FL3 to try and keep the green population off the axis. Have
you ever run GFP along with a red dye? Then you really do need to compensate
FL1 from FL3. Good luck.
David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center
---------------------- Forwarded by David McFarland/VUMC/Vanderbilt on 12/28/99
03:13 PM ---------------------------
Bunny Cotleur <bcotleur@ohio.net> on 12/27/99 04:59:31 PM
Please respond to bcotleur@ohio.net
(Embedded image moved to file: pic15454.pcx)From:(Embedded image moved to
file: pic06537.pcx)Bunny Cotleur <bcotleur@ohio.net> on 12/27/99 04:59 PM
To: Cytometry Mailing List
<cytometry@flowcyt.cyto.purdue.edu>
cc: (bcc: David McFarland/VUMC/Vanderbilt)
Subject: I got them compensation blues...
Flow masters-
I am having a bit of a dilemma. I haven't done any 3-color work in many
many years. And frankly, I don't recollect this issue cropping up
before. What I am trying to do is fairly simple. I am multi- staining
human lymphocytes for various chemokine receptors simultaneous with CD4
and CD8. I have the following mixes (set up by someone in the labs,
who's work I am extending):
CD4-PERCP/CD8-FITC/CHEMOKINE*-PE
CD4-PERCP/CD8-PE/CHEMOKINE*-FITC
(I am staining for several chemokine receptors, the exact ones not
important here).
Now here is my simple problem that is dragging me down into absolute
madness. When I PROPERLY compensate Fl1/Fl2
(I say properly,as I have followed Mario's web instructions TO THE
LETTER. Except for all the crazy math that is making my head spin :-)
).... I get frustrated. When FICT/PE are comp'd fine, then PE is
smack in quadrant 2 looking at PE vs Fl3. So, OK- I comp FL2/Fl3.
Picture perfect. Go back to Fl1 vs Fl3. AAARRRGGHHH! Green is now
way overcomp'd to the X axis. Let's just get scientific and say really
SQUOOSHED down. I have been going back and for with this for days.
Different sample sources. Titering down the Ab's. I call BD and they
say maybe my instrument is "too sensitive". HUH??? Is that possible?
I thought that was IDEAL. IN the meantime, I also note that %CD8 FITC
is NOT equal to %CD8PE in the same sample. (Both sources=BD, both
used as they recommend. Both also titred down to 2ul, no difference).
Now I'm starting to feel that I need to surrender my "License to Flow"
due to acute ignorance.
MAYDAY!
(***white flag is flapping in the breeze**)
--
Bunny Cotleur
Cleveland Clinic Foundation
Neurosciences / NC30
216-444-1164
******************************************************************
When you do something, you should burn yourself completely, like a good
bonfire, leaving no trace of yourself.
(Shunryu Suzuki)
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