Thank you for addressing this. I was misled by the promise of PE-CY7 as a solution for non specific binding of CY5 to cells of monocytic lineage. Not only does it bind to these cells the same way that PE-CY5 does, it is dimmer than PerCP. After consulting with some experts I found out that there were two problems with this fluorochrome. The first is that my FACScan is not set up to "see" the maximum emission from the CY7. The second is that the difference between the emission of PE and the excitation of the CY7 is far from optimal resulting in the CY7 signal being very dim. Margaret >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. >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Margaret M. Tropea Critical Care Medicine Department National Institutes of Health (ph) 301-496-7752 (fax) 301-480-3389 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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