Yes, I would also like to know how to deal with compensating FL1 out of FL3 in
particular. Take for instance a 3 color experiment with GFP, PE and 7AAD. The
GFP does overlap into FL3 where FITC wouldn't be much of a problem. It doesn't
make any sense (to me anyway) that FL1 is compensated out of FL3 by way of FL2-
%FL1 and FL3-%FL2, which is what BD proposes. Can someone enlighten me?
David McFarland
Howard Hughes Medical Institute
Vanderbilt University Medical Center
Flow Cytometry Facility
_______________________________________________________________________________
Subject: FL3 compensation problem with BD FACSsort
From: hildh000@goofy.zdv.Uni-Mainz.de (Jörg Hildmann) at +inet
Date: 10/05/1997 5:35 AM
Hello everyone,
I was glad that I found latex particles with are coupled with a fluorochrome
mixture which absorb at 488nm and send out 685nm (Molecular Probes,
Transfluorospheres 488/685nm, 1=B5m).
But my happiness ended when I tried to measure a triple fluorescent
population of cells labelled which FITC, PE and these 488/685nm beads. These
beads are very well labelled that one particle gives about 3 logs of
fluorescence. In FL2 early 2,5 logs are measured and can be compensated by
10% FL3->FL2. But there are nearly 1,5 logs left in the FL1 (i think that
the excision fluorochrome doesn't give all its absorbed energy to the
emission fluorochrome and emits itself) where I get a really big number of
FL1 negative and 1+ FL3 positive populations (and also each combination of
them).
The FACSsort with CellQuest 1.2 allows only to compensate between FL1/FL2 or
FL2/FL3 - not FL3->FL1 or FL1->FL3. Compensation of FL3->FL1 over
FL3->FL2->FL1 destroys completely FL1 by a strong FL2 signal or no
compensation results in multiple FL1-/FL3+, FL1+/FL3- and FL1+/FL3+
populations which make a good analysis very hard and presentation of results
nearly impossible.
I think there are only two possibilities for me! {1} Who knows how I can
overcome these compensation problem by software (Upgrade on CellQuest 3.1
doesn't solve the problem because FL3->FL1 isn't implemented yet) or
hardware (is there any other detection filter combination available to
prevent huge FL3->FL1 influence). {2} Another type of polystyrene particles
available which haven=92t these FL3-> FL1 compensation problem (like the BD
calibrate beads 2=B5m with PerCP label - PerCP is a monomeric protein and=
has
nearly no FL3-> FL1 activity - but particles have wrong diameter).
Thanks for all help and good luck for your experiments
Joerg Hildmann
-----------------------------------
Joerg Hildmann
Institut fuer Immunologie
Universitaet Mainz
Obere Zahlbacher Strasse 67
55313 Mainz - Germany
Telefon: +49/6131/17-3988 oder 2119
Fax: +49/6131/3-5688
E-Mail: HILDH000@MAIL.UNI-MAINZ.DE
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