APC-Cy7 and PE-Cy7 compensation

Disterer, Petra (Medsch Royal Free/Medicine/Hepatology) p.disterer at medsch.ucl.ac.uk
Mon Nov 17 05:21:41 EST 2008


Dear all,
Thank you all very much for clearing up my confusion over the compensation issue. I had
not considered that excitation by different lasers would permit distinction of the
resulting emission spectra based on optical pathways.
This is why I am on this list...
Thanks again
Petra Disterer

	-----Original Message----- 
	From: Rui Gardner [mailto:ruig at igc.gulbenkian.pt] 
	Sent: Thu 13/11/2008 22:43 
	To: Cytometry Mailing List 
	Cc: 
	Subject: Re: APC-Cy7 and PE-Cy7 compensation
	Because they're detected in two different channels. Most standard flow cytometers
have their lasers separated spatially so that the fluorescence of, say, two fluorochromes
with a close emission spectrum but excited by two different lasers, can be detected
separately, i.e., through two different optical paths. PE-Cy7 is generally excited by a
blue 488nm laser (though a green or better, a yellow laser would be best), and APC-Cy7 is
usually excited with a red 635nm laser. At the end of each optical path, you can then use
the same filter range for both. 
	Beautiful machines, aren't they!? ;-)
	Rui.
	_____________________________
	Rui Gardner
	Flow Cytometry Lab Manager
	Instituto Gulbenkian de Ciência
	Apart. 14, PT-2781-901 Oeiras
	PORTUGAL
	Email: ruig at igc.gulbenkian.pt
	Disterer, Petra (Medsch Royal Free/Medicine/Hepatology) wrote: 

		Dear all,
		Maybe my understanding of the idea of tandem dyes is wrong, but don't
APC-C7 and PE-Cy7
		have the same emission spectrum seeing as Cy7 is the acceptor dye in
both? So, how can
		you compensate between APC-C7 and PE-Cy7?
		Enlightenment would be highly appreciated.
		Petra Disterer
		UCL Medical School, UK
		________________________________
		From: Guzik, Lynda [mailto:GuziLJ at ccm.upmc.edu]
		Sent: Fri 11/7/2008 16:39
		To: cyto-inbox
		Subject: APC-Cy7 and PE-Cy7 compensation
		Hello All,
		I have a researcher who would like to do 6color flow using FITC, PE, PI,
APC, APC-Cy7,
		and PE-Cy7.  We are using a FACSVantage DiVa with a 488nm, 635nm and UV
lasers.  We are
		having problems with compensation between the APC-Cy7 and PE-Cy7
channels.  Using single
		color positive controls and the DiVa algorithm, the compensation looks
fine.  However,
		when we put APC-Cy7 and PE-Cy7 antibodies in the same tube, the plots
look like there is
		extreme overcompensation of PE-C7 against the APC-Cy7 channel.	I've
attached a couple of
		plots to illustrate.  I am able to correct for this, but wonder if I
should be.  And I
		fail to understand why the single color controls look fine but have a
problem with the
		dual stained tube.  Any input would be appreciated.
		Thank you,
		Lynda Guzik
		McGowan Institute - Lagasse Labs
		Rm 520A - Bridgeside Point Bldg
		100 Technology Dr - Suite 200
		Pittsburgh, PA 15219
		412-235-5213
		guzilj at upmc.edu<mailto:lguzilj at upmc.edu> <mailto:lguzilj at upmc.edu>  or
lyg1 at pitt.edu<mailto:lyg1 at pitt.edu> <mailto:lyg1 at pitt.edu> 






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