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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