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Dear Peter Chapple,
I have asked John Wotherspoon from BD Australia to follow up with you directly
on your FACSCalibur queries. But for all other interested parties, Tom Frey, a
scientist in our Research group that worked on the FACSCalibur development adds
these comments.
"The 3-color Calibur uses a 650LP, the 4-color uses 670LP. The maximum emission
of a PE/Cy5 is within either of those filter ranges. PE/Texas Red is shorter and
you may only pick up the tail (although experience suggests that works well
enough in most cases.
BD recommends PerCP in order to avoid some of the compensation problems
that can occur with PE/Cy5 conjugates. They always emit some PE fluorescence
(although often not much with a good conjugate). The amount of PE fluorescence
can vary from conjugate to conjugate or from vendor to vendor. Also, it is
fairly well known that exposure to light can bleach the Cy5 fluorochrome and
lead to more emission in the PE channel. Labs that are less careful about light
exposure might see this as an increase in FL2 over the lifetime of the bottle of
reagent."
As for the FACSCalibur manual, the Chapter on FL4 does have a diagram showing
the optical layout and the filters that are used in FL1-FL4. If for some reason
any FACSCalibur customer did not receive the FL4 chapter when they received
their FL4 upgrade, let us know and we will get one right out to you. Also, if
you have any concerns regarding the documentation of the FACSCalibur, we would
love to hear them directly, so we can make it better to meet your needs. Feel
free to contact me at BD directly with comments or concerns and I will forward
them on. Thanks.
Angela K. Schultz
BDIS Marketing/FACSCalibur (Development) Program Manager
email: angela_schultz@bdis.com
_______________________________________________________________________________
Subject: PE-Cy5 on Facscalibur
From: Peter Chapple <peterc@petermac.unimelb.edu.au> at INTERNET
Date: 6/18/97 12:33 PM
Greatings all,
I have been looking into the economics of our flow setup and discovered that I
can buy PECy5 anti CD45 for much cheaper than I currently buy PerCP anti CD45. I
proceeded to make some three color CD3/4/45 and 3/4/45 PBL's to do some
compensation with and found that I got into real trouble :(
Did something drastic go wrong with the labeling of the PBL's ??, or is there
something I need to know about PE-Cy5 on a Calibur (ie: has anyone else done
this easily/with difficulty ??)
One problem is that I have very little quality information on the Calibur - the
manual seems to make no mention of the of exactly which mirrors/filters the
Calibur uses ... does it use a 650LP filter on the FL3 detector the same as a
FacsScan does ?? (if that's the case then I'm only just picking up the *tail* of
the PE-Cy5 emission)
Peter Chapple
Melbourne AUSTRALIA
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From: Peter Chapple <peterc@petermac.unimelb.edu.au>
To: cyto-inbox
Subject: PE-Cy5 on Facscalibur
Date: Wed, 18 Jun 1997 12:33:47 +1000
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web