[Cytometry] Compensation, Cy5PE, and APC

Geza Paukovics paukovic at burnet.edu.au
Thu Feb 6 20:21:42 EST 2014

Dear Mario and Ian,

Mario I totally agree with your statements, while it can be tricky people
can in fact set a panel of PE, PE-Tx, PECy5, PECy5.5, PECy7, APC, AF700 and
APCCy7 IF they need to and their tandem fluorophores are "fresh", have good
integrity and are well taken care of (nominal if any tandem degradation
I also totally agree with Ian, that sometimes core managers recommend
fluorophores to eliminate potential problems, problems that can be avoided
looking at more "idle" clients. Not all labs are well funded, not all labs
are always busy, we know research can be cyclical, with periods of heavy
flow work = antibodies/fluorophores do not sit around. We all, as core
managers should know there is a number of important points to consider when
asked to recommend fluorophores. Target antigen expression is but one. Most
core managers also know the load of work each unit has or undertakes, staff
numbers etc... how quickly is the Ab-fluorophore going to be utilized in a
project is another important point. If you have clients with high work flow
rates with numerous staff, there is a good chance Ab usage will also be
high = Ab will be used up quickly, hence reducing the chance of tandem dye
degradation. How staff handle the Ab in a lab is also a factor. Sometimes
staff or students can be careless leaving an antibody uncapped under
fluorescent light or in a hood etc... this will especially affect tandem
conjugates. In a pressure environment where every cent and dollar needs to
be utilized, some people use "older" antibodies/tandem conjugates as well
(not out of date obviously). By some core manager recommending PECy5.5 (I
believe more stable over time) instead of PECy5 with APC, I think we're
helping our users to get the most "bang" out of their buck so to speak.
Some horses, are for some courses.
Happy Flowing all

On Fri, Feb 7, 2014 at 1:21 AM, Ian Dimmick <ian.dimmick at newcastle.ac.uk>wrote:

> 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
> 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
> UK
> 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!)
> >
> >cheers,
> >
> >mr
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*Geza Paukovics*
AMREP Flow Cytometry Core Facility Manager
AMREP Senior Flow Cytometrist
Burnet: Corporate Support Services - Laboratory Services
VHPF Flow Cytometry Program Group
Sidney Myer Fund Flow Cytometry Unit
Monash: Monash Central School of Medicine (AMREP)

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