IFN far red tandem conjugates: responses

Prussin, Calman (NIH/NIAID) CPRUSSIN at niaid.nih.gov
Wed May 12 10:57:20 EST 2004

Below are the two responses I got. We ended up using PE/Cy7 with good
results. I also see now that PHGM has anti-hu IFN in Alexa 700. Regarding
comment #2 & compensation: PharMingen and likely others as well, have
compensation beads (compbeads) that can be used for compensating tandem
conjugates that recognize dim or rare markers.	  Calman
1. APC-Cy7 IFNg from BD works pretty well for me.   I am using MoFlo with 
a minor modification to bring up a signal generated by a Red Diode 
laser (620-690).  It is not as bright as PE or FITC-anti-IFNg but a 
proportion is very much within a statistical range.

2. We have pharmingen IFNg PECy7, and it is very good.	My only concern is
with compensation.  There is lot to lot variability on compensation of the
acceptor (e.g. cy5) against the donor (PE).  If you are dealing with tiny
populations, such as allergen specific responses, you might not have enough
cells to compensate.  Gamma is not that much of a problem for obvious
reasons.  Of course, biotin-avidin is a way to go as well, and since you can
use zenon, which changed your life, it is an option. 

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> ----------
> From: 	Prussin, Calman (NIH/NIAID)
> Sent: 	Wednesday, April 28, 2004 15:00
> To:	Cytometry Mailing List
> Subject:	IFN  far red tandem conjugates & a question for Mario
> Cytokindred,
> After some initial negative experiences long ago, we have generally
> limited
> ourselves to using non-tandem conjugates for our anti-cytokine mAbs used
> for
> ICCS. I'd like to revisit the issue and try some anti-human IFN-g tandem
> conjugates, particularly those emitting in the far red, such as PE-Cy7 and
> APC-Cy7.
> I am requesting feedback from individual investigators regarding
> particular
> products they have used successfully. All replies I get will be considered
> for non-confidential unless you specify otherwise.
> Company reps are welcome to email me directly to inform me of their
> product's availability, but please do not reply to the list.	
> Mario: A question I have always wanted to ask you. My instinct is to refer
> to a tandem with the excited dye noted first and the emitting dye noted
> second (e.g. PE/Cy5). I use this order because it is in the order in which
> the light energy interacts with the tandem (first excited, then emitted).
> Mario, you always use the opposite convention (e.g. Cy5/PE) and must have
> a
> good reason for doing so, what it it?

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