Complex Compensation :(

rozenkov@netscape.net rozenkov at netscape.net
Sun Apr 23 03:02:48 EDT 2006


Dear David,
For your question A, you can refer to some previous discussions on this list, for example
2005's archived discussions in September and another one in October-November, the latter
was held under the title "Algorithm for manually setting multicolor compensation".
For your question B, it is not enough for correct compensation to use a fluorochrome of
the same color (similar emission wavelength) instead of the stain that will be used in
real experiments. Different fluorescent substances, even though they have the same color,
i.e. similar emission peak wavelength, will have different shapes of emission spectrum
histograms, hence different overlap with other channels, and will require different
compensation. Ideally, you should use the same fluorochrome labeled beads or particles
that you have. You will probably hear from other respondents as well that even the same
fluorochromes from different companies may differ, and even different batches
sometimes...
Regards.
Vladislav Rozenkov, MD, PhD
-----Original Message-----
From: David Alvarez <alvared at mcmaster.ca>
To: cyto-inbox
Sent: Thu, 20 Apr 2006 13:48:35 -0400
Subject: Complex Compensation :(


Good-day.
Having worked with LSRII for some time now, I am starting to get a little bit frustrated
with compensating non-traditional fluorochromes/dyes. My previous experiences had been
with directly-conjugated Abs, which worked like a charm for T cell immunophenotyping.
Nowadays I study murine DCs and monocytes, and I am using fluorescent microbeads and dyes
like TRITC. Not sure, if there is anyone who has experience with these particular
fluorochromes, but I'm hoping to get a little advice with compensation.
Normally I perform automatic compensation. Which is soooo easy!! However, some of these
TRITC or microbead-positive cells are too rare to autocompensate! Therefore, I have
switched to manual compensation. This has NOT been easy. I think what I need (ultimately)
is a clear algorithm to perform every time I compensate the LSRII. Can you help?
Several items I should mention:
[A]    The typical colors I use in a single experiment are as follows:
	-green latex beads (FITC channel)
	-TRITC dye (PE channel)
	-traditional colors (PerCP, Pe-Cy7, APC, & APC-Cy7)
[B]    I have been compensating the LSRII with single stained controls. (When it comes to
microbeads or latex particles I believe I should be using those, rather than
directly-conjugated Abs of the same wavelength.)
Please, please advise.
dAVID
David Alvarez, PhD
Gene and Cell Medicine
MSSM
NY,NY


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