[Cytometry] Compensation, Cy5PE, and APC

Ian Dimmick 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 



Ian Dimmick 
Flow  Cytometry Core Facility Manager
Newcastle Upon Tyne University 
Faculty of Medical Sciences 
Bioscience Centre (West Wing)
International Centre for life 
Newcastle Upon Tyne 
NE1 3BZ 
Ian.Dimmick at ncl.ac.uk 
CFL Office                           Tel   0044 191 2418831 
CFL Laboratory                Tel   0044 191 241 8825
Med. School office           Tel   0044 191 222 7155
Med School Laboratory Tel   0044 191 222 7079
Mobile number                Tel    0044 7970344823

Website http://www.ncl.ac.uk/fccf
Booking calendar http://flowcytometry.calendarhost.com
Core facility Forms send to  fccf at newcastle.ac.uk

>-----Original Message-----
>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!)
>Cytometry mailing list
>Cytometry at lists.purdue.edu
>Search the list archive at  http://tinyurl.com/cytometry

More information about the Cytometry mailing list