Jeff,
Pardon me if this is too obvious, but I would also suggest tweaking the PE
signal by slightly adjusting the dichroic between FITC (FL1) and PE (FL2).
My thought is that it MUST be something pretty obvious though, because PE
(FL2) is one signal that I've never had a problem with! And as far as I
know, the 575/26 has always been in place.
And Joe, FYI the 575/26 is listed as the standard PE (FL2) filter for all
configurations listed in Appendix B (pp308-312) of the FACSDiVa User's
Guide. I for one had no idea that this was intended for Texas Red and
that a 585/42 was suggested for PE. (I just took a quick look and I do
indeed have a 585/42).
Cheers,
Dave
David McFarland
GlaxoSmithKline
----- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 29-May-2002 08:46
-----
JTrotter@PharMingen.com
24-May-2002 13:11
To: "Cytometry Mailing List"
cc:
Subject: Re: How to enhance PE signal?
Jeff,
Usually, when this occurs, the Vantage user has PE band pass filter
designed for use with Texas Red in the FL2 channel (a 575/26) , and the
Calibur uses a 585/42. Have a look and replace the filter if necessary,
you
should also have a 585/42 for the Vantage in your kit that will let in a
lot more PE signal. You didn't mention what laser power you use, 100 - 200
mW on the sorter?
Joe
"Jeffrey Rice" <rice@aecom.yu.edu> on 05/23/2002 07:05:47 PM
Please respond to rice@aecom.yu.edu
To: cyto-inbox
cc:
Subject: How to enhance PE signal?
Hello,
I'm running into a problem here with my sorting. I've got a
peptide-tetramer
using biotinylated
peptide with SA-PE. On our Calibur, I get between half a log and a log
difference
between my negatives
and positives, but on our Vantage+DiVa I lose most of that. I really need
to enhance
the PE signal from
my PE positives to improve my sort.
Now, I know things like Oregon Green labeled anti-FITC exist. Do any
FL2-labeled anti-PE
reagents exist? Or, FL4-labeled anti-APC would be OK too.
I've tried switching fluorochromes... I get good labeling on the
Calibur with
a direct labeling of the
protein using Alexa Fluor 647 (FL4), but again I lose the separation
between my
populations when I try to
sort on the Vantage. I could use a brighter SA-fluorochrome conjugate for
my tetramers,
or a brigher
fluorochome to label the protein. But I would think that using SA-PE or
SA-APC for
tetramers, or Alexa
Fluor 647 for direct labeling, are among the brightest available.
Any suggestions would be greatly appreciated, as I'm fairly well
stuck
at
the moment.
Jeff
----
Jeffrey Rice
rice@aecom.yu.edu
Diamond Lab
Microbiology and Immunology
Albert Einstein College of Medicine
Bronx, NY
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