Hello Andy,
We have a MoFlo but did notice something similar with PE-Cy7. We have
a 3 laser bench. Our first laser is a HeNe, 2nd one is kept at 488nm
and the third one we convert to various wavelengths. I had the 3rd
laser set at 514nm and compared the signal for PE-Cy7 (785/50) at
488nm or at 514nm. We only had positive staining if the signal was
collected off the 514nm (3rd) laser path. We saw nothing from the 488
(2nd)laser. At first we thought maybe it was the difference in
exciting PE from the 514 vs 488. So I move the 488 laser into the 3rd
laser path and we did get a positive signal. Also, if I moved the
514nm laser into the 2nd laser path we had positive signal in the PMT
that gave no signal with 488nm excitation of PE-Cy7. Thus, it wasn't
the difference in the laser wavelength or in the 2 PMTs we were
comparing. What it turned out to be was that I had a blocking filter
in front of the laser 2 PMT stack to block any stray 514 light coming
in to the 488 pmts stack when both lasers are on. It is a 521LP
filter. When this was removed, we had positive signal for PE-Cy7 from
the 2nd laser (488). So even though this is a long pass filter it
blocks signal in the 785/50 range.
Possibly you have a bogus filter in your setup. Good luck
Lucy Brown
Beckman Research Institute/City of Hope
626-359-8111 X3306
lbrown@coh.org
____________________Reply Separator____________________
Subject: Decreased signal in FL3 with SE upgrade
Author: "MORSCHAUSER, ANDREW" <ANDREW.MORSCHAUSER@spcorp.com>
Date: 12/19/00 11:04 AM
This message is directed towards FACSVantage users that have
upgraded their
machine from a FACSVantage to a FACSVantageSE w/Turbosort. I
am
experiencing a
decreased sensitivity in FL-3. When comparing alignment
particles (cRBC,
Alignflow beads, Calibrite beads) between a FACSVantage,
FACSVantageSE w/TSO
and
a FACSCalibur, results have shown the FACSVantage SE signal to
be
significantly
lower than the Vantage or Calibur. Furthermore, when looking
at splenocytes
stained with CD4 PE-Cy7 (787/40 filter), there is no
discrimination between
negative/unstained cells vs. stained cells. The signals on the
FACSVantage
and
Calibur are nearing the third decade while the FACSVantageSE
signal was
negative
with log amps and voltages greater than 900V. I tried
increasing the log
offset
as far as it will go and found little if any improvement in the
signal.
Boards have been replaced and new PMTs in FL3 have done nothing
to remedy
the
problem. BD has been very responsive to my concerns, but the
problem is
still
present and I was wondering if anyone else has experienced
similar problems
with
the extra mirror and different optical path for a 5-color
FACSVantage SE.
Any
comments or suggestions would be greatly appreciated.
Andy Morschauser
Schering-Plough Research Institute
Department of Immunology
2015 Galloping Hill Road
K15-3-3945
Kenilworth, NJ 07033
908-740-3089
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