[Cytometry] Compensation, Cy5PE, and APC
ian.dimmick at newcastle.ac.uk
Thu Feb 6 09:21:31 EST 2014
Yes Mario , completely agree , however I think you have summed up the practical approach , that also avoids large dye spread , and reduction in sensitivity because of it , WHY BOTHER, when there are so many other alternates that you can go for , most popular being PEcy5.5
I think those of us working in large core facilities very often consider what we can do (your last example ">(It might be noteworthy that we have used all of Cy5PE, Cy5.5PE, APC,") and what we should advise to end users that are perhaps utilising flow Cytometry , and not using it as their major interest or application .
So don't knock people for being practical and working within the parameters of their core facilities , what works for one ..........................
This is my weekly rant
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>From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-
>bounces at lists.purdue.edu] On Behalf Of Mario Roederer
>Sent: 06 February 2014 13:44
>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!)
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