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 Received: from flowcyt.cyto.purdue.edu (128.210.60.31) by cc.bdis.com with SMTP (IMA Internet Exchange 2.1 Enterprise) id 0000998D; Wed, 18 Jun 97 12:05:10 -0700 Received: by flowcyt.cyto.purdue.edu (940816.SGI.8.6.9/930416.SGI.AUTO) for cyto-sendout id MAA21015; Wed, 18 Jun 1997 12:30:17 -0500 Received: from bastion.petermac.unimelb.edu.au by flowcyt.cyto.purdue.edu via ESMTP (940816.SGI.8.6.9/930416.SGI.AUTO) for <cytometry@flowcyt.cyto.purdue.edu> id VAA18792; Tue, 17 Jun 1997 21:49:15 -0500 Received: (from mail@localhost) by bastion.petermac.unimelb.edu.au (8.6.8/8.6.10) id MAA03573 for <cytometry@flowcyt.cyto.purdue.edu>; Wed, 18 Jun 1997 12:57:28 +1000 Received: from server.petermac.unimelb.edu.au(172.20.7.15) by bastion.petermac.unimelb.edu.au via smap (V1.3) id sma003549; Wed Jun 18 12:56:43 1997 Received: from path5.petermac.unimelb.edu.au ([172.20.10.223]) by server.petermac.unimelb.edu.au (8.6.8/8.6.10) with SMTP id MAA25429 for <cytometry@flowcyt.cyto.purdue.edu>; Wed, 18 Jun 1997 12:45:58 +1000 Received: by path5.petermac.unimelb.edu.au with Microsoft Mail id <01BC7BE3.DF91D3C0@path5.petermac.unimelb.edu.au>; Wed, 18 Jun 1997 12:33:49 +1000 Message-ID: <01BC7BE3.DF91D3C0@path5.petermac.unimelb.edu.au> 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 MIME-Version: 1.0 Content-Type: text/plain; charset="us-ascii" Content-Transfer-Encoding: quoted-printable
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