[Cytometry] Compensation, Cy5PE, and APC

Alvaro Luiz Bertho bertho at ioc.fiocruz.br
Thu Feb 6 11:27:09 EST 2014

Dear Mario,
Very good explanation and necessary!!! 
We also used PECy5 and APC  and others fluorochromes at the same time and we got nice results, at least in Cyan ADP and MoFlo Astrios flow cytometers. However, I will make mine, your words: "I'm not saying it's easy, but I am saying it's not impossible!"
Best regards
Alvaro Luiz Bertho, PhDPrincipal InvestigatorHead of Flow Cytometry Core FacilityOswaldo Cruz Institute - FIOCRUZRio de Jan

> From: roederer at drmr.com
> Date: Thu, 6 Feb 2014 08:43:38 -0500
> To: cytometry at lists.purdue.edu
> 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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