Re: PerCP vs PerCPCy5.5 (or PE-Cy5.5) on FACS Aria or LSR II

>We all know that a few years ago, BD introduced PerCP-Cy5.5 tandems, 
>with the claim that they perform better on the jet-in-air sorters; 
>it is not my intent to discuss that.
>
>Instead, I want to discuss which reagents are preferred on the Aria 
>(or LSR-II) equipped with an octagon on the 488 laser and a trigon 
>on the 633 laser. Specifically, the list I'd like us to consider is 
>PerCP, PerCP-Cy5.5, and PE-Cy5.5. For the moment, I'd also like to 
>exclude from the conversation attempts to simultaneously use a Cy5.5 
>tandem between a Cy5 and a Cy7 tandem (unless some insists, for a 
>REALLY good reason).
>
>My first order expectation is that of the three labels (PerCP, 
>PerCP-Cy5.5, or PE-Cy5.5), the preferred one on the Aria would be 
>PerCP, for one simple reason -- the Cy5.5 moiety on the tandems will 
>be excited by the red laser and will therefore require large 
>cross-beam compensation in the channel that we are currently using 
>Alexa680 or Alexa700. In contrast, the required crossbeam 
>compensation for PerCP will be substantially less.
>
>Any thoughts?

Hi John -

Since you are using digital electronics (on an Aria) whether or not 
the compensations needed are "cross-beam" or come from the same laser 
is immaterial. It is all handled in the same manner by the same 
computations.

Unfortunately there is a good reason for considering other colors. 
And that is because, depending upon whic ones you use, there may be 
spectral overlap into the PerCp measurement channel. This will be 
especially true of PE-TR, and is relevant because out of the three 
dyes considered, PerCp is the dimmest, followed by PerCp-Cy5.5, 
followed by PE-Cy5.5. And of course the emission spectra of PerCP and 
PE-Cy5 are too close to be able to independently measure both dyes, 
while the Cy5.5 tandems can be measured along with PE-Cy5.

The Cy5.5 signal does show up strongly in the red excited channels, 
but the PerCp, although less, is not insignificant either. So you 
have a balancing act to do. You will obtain better results with the 
red laser excited dyes by using PerCP rather than a tandem Cy5.5. 
However you will obtain worse results on that primary channel with 
PerCP. And, of course, the PE tandems will show up on a number of 
other blue laser channels, while PerCp only has overlap with the 
longer wavelength emissions (PE-Cy7). Thus, to me, the "correct" 
answer to your question is: it depends upon what your are trying to 
measure. It may make no difference at all which fluorochrome you use, 
or it may be quite significant.

If you have an Aria now with a low power 488 nm laser, you also need 
to consider, if the panel you are designing will have a long 
lifetime, that at some point in the future you may want to upgrade to 
a higher power one. A 100 mW blue laser is a drop-in replacement and 
provides substantial signal increases compared to the stock low-power 
blue laser. We will be presenting some date demonstrating this at 
ISAC.

Marty


>
>John
>
>
>--
>John D. Altman
>Associate Professor
>Emory Vaccine Research Center
>954 Gatewood Road
>Atlanta, GA 30329
>
>jaltman@rmy.emory.edu
>http://microbiology.emory.edu/altman
>Office Phone: (404) 727-5981
>Lab Phone: (404) 727-8914
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