Hi Mario A puristic question: Is it PE-Cy7 or Cy7-PE. I would use the first as the Cy7 piggybacked on the PE which is also the direction of energy transfer. Also, would you be in a position to make up a table of names the companies use for the various base conjugates, their emission / excitation properties. Or has howard perhaps already one in preparation similar to the one in his book with the various dyes and the major lines from the available sources? Thanks in advance Gerhard -----Original Message----- From: Mario Roederer [SMTP:Roederer@drmr.com] Sent: Tuesday, November 30, 1999 7:13 PM To: Cytometry Mailing List Subject: Re: 4 colours on FACS Calibur In contrast to the somewhat misleading commercialism of this post, Cy7PE was NOT specifically formulated to get around this problem (nor was it developed by Caltag, as seems to be the implication). Aaron Kantor and I (in collaboration with Alan Waggoner) developed this tandem dye at Stanford to provide us with what was then our sixth FACS color. It does have the nice advantage that it can be used in concert with APC on diode lasers with less compensation than Cy5PE. However, the assertion that it is not excited by the diode laser is false; Cy7 can be indeed be excited by the diode laser. The big reason for the relatively low compensation between FL3 and FL4 is because the Cy7 emission is shifted about 30 nm to the red from APC, so it doesn't fall into the APC filter as much. Of course, all of these values depend heavily on the precise excitation line and emission filters that you use. Other alternatives to four color systems include using Cy5PE together with Cy7APC (available from PharMingen, and I think from Caltag as well), Cy5PE together with Cy5.5APC, or Cy5.5PE or Cy5.5PerCP (from BD) together with APC or Cy7APC. (i.e., there are lots of possibilities; the availability of such conjugates will become far more prevalent as the multi-color instruments infiltrate our ). I would remind anyone who uses tandems like Cy7PE that nearly every different conjugate, even purchased from the same company, may require a different compensation setting. This makes proper compensation on the instrument for multiple staining panels nearly impossible; currently, the best solution is to use software that can manage multiple different compensation settings assigned to specific panels (and in this case, it would be completely accurate to say that "FlowJo (<http://www.treestar.com/flowjo>) was specifically formulated at Stanford to get around this problem"). mr At 5:28 PM -0500 11/29/99, Karenrt@AOL.COM wrote: >There is another alternative to PerCP for 4-color analysis on the >FACSCalibur. PE-Cy7 is available from Caltag Laboratories and was formulated >specifically to get around this problem. The diode laser on the FACSCalibur >does excite Cy5, causing compensation problems when using it with APC. Cy7, >however, is not excited by the red diode. There is very little if any >compensation between FL3 and FL4 with this conjugate, and about 30% between >FL2 and FL3, For a list of antibodies conjugated to this fluorochrome, check >our Website at www.caltag.com. > >Karen R. Tamul, MS, MT(ASCP)SI >Mid-Atlantic Territory Manager >Caltag Laboratories Inc.
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