From: Mario Roederer (Roederer@Beadle.Stanford.EDU)
Date: Thu Nov 14 1996 - 21:25:30 EST
Recent discussion about the 4th color on FACSCalibur prompts me to point (again) that, as Angela Schultz points out, there are many options. These are based on the recently developed Cy7 tandems of both PE and APC. While Tom Frey is correct that Cy7 is not efficiently excited at 635 nm, my guess is that Tom Delohery was talking about the Cy7 tandems. Cy7 tandems are nice, bright dyes. Their "useful" brightness is approximately 6-fold as good as fluorescein (whereas PE and APC are about 8-12 times the brightness of FITC, and Cy5PE is about 20 times as bright). Note that the Cy7 tandems can be made even brighter when using them with specific applications. I'm sorry, but I don't have information about the relative brightness of PerCP in these systems. Also note that I made these measurements on a jet-in-air configuration; the FACScan has even more sensitivity for the phycobiliproteins and their relative brightnesses increase. First of all: what about the Cy7 tandems? They are not particularly difficult to make (see http://cmgm.stanford.edu/~roederer/abcon). I am trying to get some manufacturers to actually market them as well; possibly as a kit (ready to conjugate to antibody of your choice!). If you are interested in Cy7 tandems, send me EMail and I will keep a list for future reference. So what are the 4 color possibilities? (1), there is a 4-color one laser combination that works very well: FITC, PE, Cy5PE, and Cy7PE. I don't know if the FACSCalibur can be configured for this combination or not. (2), the 4-color one laser combination of FITC, PE, PerCP, and Cy7PE should work as well on the low power systems. The priniciple compensation needed for these combinations is between Cy7PE and PE. For two laser excitation, the best combinations would be: (3) FITC, PE, PerCP, and APC. Works well because of the little compensation required between PerCP and APC. (4) FITC, PE, Cy5PE, and APC. Because of the efficient Cy5 excitation by the second laser, this probably works only well for systems where (as was previously noted), Cy5PE and APC are used to measured antigens expressed on distinct subsets (for instance, CD3 and CD19). (5) FITC, PE, Cy7PE, and APC. As Tom pointed out, Cy7 is not efficiently excited by the second laser. Thus, this combination should work very nicely. (6) FITC, PE, Cy5PE, and Cy7APC. While the second laser is exciting Cy5, the emission will be largely ignored since the second laser channel will be "tuned" to Cy7--requiring little compensation. (7) FITC, PE, Cy7PE, and Cy7APC. Less compensation than 6, but sacrificing some of the Cy5 brightness. (8) FITC, PE, PerCP, and Cy7APC. Not much benefit over #4. The best combinations will still have to be determined empirically (by best I mean the greatest combination of bright reagents). I would speculate that #5 and #6 would probably end up the best. By the way, the absolute best would probably be the combination of PE, Cy5PE, Cy7PE, and Cy7APC, and using a Green HeNe and a Red HeNe for excitation! I can't wait for that system to be put out! mr
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