>Andy,
>
>I'm head of the group tha developed PharRed at PharMingen. Could you please
>tell me exactly what you mean by decoupling? Given the overlap of PharRed
>with
>APC you should see a population in the APC channel when running a single color
>PharRed sample.
>
>So far, in inhouse tests we have not seen a breakdown of PharRed in either
>accelerated stability tests or in real time over a year. I would would be
>very
>interested in knowing what problems you have had so we can try and solve them.
>PharRed provides an important extra color and I want to make sure that it
>works
>as researchers would expect it to.
>
>Have you seen a change in the amount of compensation required for APC-%PharRed
>over time? What system (Cytometer, laser, filters etc) are you using? Let me
>know what we can do to make PharRed more usable for you. In general you
>should
>expect a fluorescence equivalent to FITC.
>
>Sincerely,
>
>Alan
>_______________________________________________________________________
>Alan M. Stall, Ph.D.
>Vice-President, Research Immunocytometry
>
>BD-PharMingen Tel: (858) 812-8843
>10975 Torreyana Rd. FAX: (858) 812-8888
>San Diego, CA 92121 E-mail: AStall@PharMingen.com
> WWW: http://www.pharmingen.com
Andy,
From what you said, it sounds to me like you're seeing
signal in the APC channel from PharRed - which is normal.
It compensates out just like PE-Cy5 does in the PE channel. Is
this what you ment, or is there other evidence that it really decoupled?
Joe
Joe Trotter
Director, Flow Cytometry
Mailstop Imm-20
The Scripps Research Institute
10666 North Torrey Pines Rd.
La Jolla, California 92037
>===============
>
>
>Hi all,
>
>I haven't used the PE-Cy7 conjugate, but have used the APC-Cy7 (PharRed)
>conjugate. I found the PharRed dye congugate to decouple. This was evident
>in single stain test controls where I saw a population in the APC channel
>when running PharRed. Has anyone else see this? How do your single test
>controls look with the PE version?
>
>Andy.
>
>
Dear Alan and Joe,
What I have seen is a very strong signal in the APC channel. We analysed
APC comparing it to PharRed on a MoFlo high speed sorter. Both dyes were
bound to the same biotin mAb to B220. Target cells were from a spleen.
Isotypes were -ve. I saw a small population in the APC channel for PharRed
only acquisition with the same intensity as the APC only acquisition. The
main population for PharRed lay on it's detector axis and could be
compensated. This suggest to me that their is some de-coupling of the
actual dye molecule that results in poor resonance energy transfer between
the donor and acceptor. We have repeated this with another batch and saw
similar results. We also checked the possibility of carryover between
samples and have ruled this out along with errors in sample preparation.
The filter sets I use are:
APC 670 DF14;
755 LP
787 DF40
Any suggestions?
Andy.
Andy Riddell
PNAC Division
MRC LMB
Hills Road
Cambridge
UK
tel: (0)1223 402218
fax: (0)1223 412178
email ar3@mrc-lmb.cam.ac.uk
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