[Cytometry] Compensation, Cy5PE, and APC

Cappella, Paolo Elia [Nervianoms] Paolo.Cappella at nervianoms.com
Thu Feb 6 10:49:33 EST 2014


Mario,

I think there are two main questions. First, why do peoples do not use
right controls? On FACSCalibur you could perform beautiful experiments
with Cy5PE-PerCPCy5.5 and APC. Of course you need be conscious regarding
reagents, coniugations and cells to be stain. I know, sometimes is a
problem of money or time. The second, is a lack of instrumental
knowledge. One is instrument performance check. People has to test each
day laser stability, PMT voltage, pressure tank and ALIGNEMENT! Again,
people has to test FL3/FL4 time delay with blu/red beads and if
necessary open the upper cover and adjust by tools... Several times I
observed difficults between FL3/4 due to bad experiment layout but a lot
of time due to fluidics&pressure and laser time delay. 
This is my short contribute.
Paolo Cappella
 
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Paolo Cappella, Dr. Biol.
Cell Assays Technologies / Flow Cytometry
Building 75 - Discovery
Nerviano Medical Sciences srl
PO Box 11 - Via Pasteur 10 
20014 Nerviano MI
phone: office +390331581367
           lab    +390331581778
           cell   +393395499154
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-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Mario Roederer
Sent: Thursday, February 06, 2014 2:44 PM
To: Purdue list
Subject: [Cytometry] Compensation, Cy5PE, and APC

It's time for my biennial rant about the misunderstandings of
compensation...

If you or someone in your facility  thinks that Cy5PE and APC  cannot be
used in the same panel, then this is for you!

I was recently approached by a user who was told by his flow core that
one could not use Cy5PE and APC at the same time because they have
completely overlapping emission spectra, and  it was not possible to do
"crossbeam compensation".  This thinking is archaic and arises from an
inability of the old machines like the FACSCalibur  to perform analog
online compensation between FL3 (Cy5PE) and FL4 (APC).   But we are in
the modern world now, and all of our compensation is digital and
software-controlled.

First: let's eliminate the term "crossbeam compensation" from the
lexicon.  There is no distinction between compensating parameters
collected from different lasers then compensating primers collected on
the same laser.

Second: In general, yes, you want to use dyes that overlap as  little as
possible. However, we have a limitation of number of dyes that are
available and sometimes we don't have a choice. The more the dyes
overlap, the more spillover-spreading you will have, and the less
sensitivity you will be able to achieve after compensation. So don't use
such combinations on antibodies that are expressed at low levels on
cells because then you won't be able to resolve them from the
background. This is part of how to build a multicolor panel; we have
published many papers on this process and how to optimize it.   But just
because dyes overlap heavily doesn't mean they can't be incorporated
into a panel.

Third: The reason that Cy5PE and APC can in fact be compensated despite
the fact that the Cy5 and APC  have nearly completely overlapping
emission spectra, is because they have distinct excitation spectra. The
amount of Cy5  emission appearing in the APC  channel can be estimated
from the Cy5PE  measurement,  and then be subtracted from the APC
measurement,  to leave only the signal coming from APC itself.  This is
what the compensation does.  Fundamentally, in order to compensate to
fluorescent molecules, all you need to have is either distinct
excitation spectra OR  emission spectra (or both).

Fourth:   using Cy5PE and APC  in the same panel is eminently doable.
As long as you are aware of the impact of spillover spreading, you will
be able to interpret the graphics just fine (but use numbers anyway).
There are perhaps better choices of  dyes:  Instead of Cy5PE, use
Cy5.5PE, Alexa680-PE, or Cy5.5PerCP if you can.  These will have lower
compensation values with APC  than does CyPE.

(It might be noteworthy that we have used all of Cy5PE, Cy5.5PE, APC,
and Cy5.5APC in the SAME panel -- with fine results.  I'm not saying
it's easy, but I am saying it's not impossible!)

cheers,

mr
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