Theory and extrapolation from excitation/emission charts are great first steps, but empirical observations also have their place in applications development. Our experience is that Cy5 works well using Rhodamine 6G dye laser excitation. Indeed, Cy5 has become our dye of choice for use with Texas Red, and we now routinely use it for 4, 5 and 6-color applications instead of APC. Our experience is that signal:noise with Cy5 is generally as good as with APC. Our most direct comparison of this issue was using a single biotinylated antibody and comparing multiple sources of Cy5-Streptavidin and APC-Streptavidin. In this kind of comparison, the very best APC-SA was less than 2-fold brighter than the very best Cy5-SA. Several APC-SA's had signal:noise ratios worse than any of the Cy5-SA's. Thus, the source/batch of the conjugated Streptavidin was more important than whether the fluorochrome was APC or Cy5. Signal:noise ratios for things like directly conjugated anti-murine CD8-Cy5 are equivalent, or better, than those we obtain with direct Fitc or PE with the same clone. Importantly, it is ridiculously simple to do your own conjugations with Cy5 using kits. CALTAG has conjugated several anti-mouse clones for us with Cy-5. These work well, we routinely (like everyday) use them and I believe CALTAG sells at least some of their clones conjugated to Cy-5 as standard products. For us, one of the major benefits of using Cy5 instead of APC is that the back cross-talk (the APC or Cy5 back into the Texas Red channel) is much, much less with Cy5 than with APC. In general, we use about 4-fold less back compensation (FL3 - FL4) with Cy5 than with APC. We generally run our dye laser at 595-600 nm, about 125mW. We use a 630/22 filter for Texas Red and a 670/14 for Cy-5. If you have a BDIS FACSTAR/VANTAGE type instrument, it is important to make sure that BD has the correct dichroic splitters and filters in your Texas Red and APC channels as they have at times been a bit confused in this area. Our experience is also that simultaneous optimization of both the APC/Cy5 channels and the tandem (Cychrome, Red670) channel requires very careful alignment. Susan Sharrow Experimental Immunology Branch National Cancer Institute 301-496-2776
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