I've had quite a lot of questions and comments about red-excited Alexa fluorochromes since my response to the APC conjugation question, so I thought I'd make a general post about the questions and summarize some of the comments. The most common is in regard to what filters we use for 633 and 660 detection. It's not entirely clear to me at this point what is best for 660, since the filters that best match the emission spectra do not give the best fluorescence detection. We're still trying to empirically determine the best filter, but right now I would suggest something around 695 to 700 bp 15 to 20. Standard APC filters (eg, 660) work pretty well for A633, although we're still tweaking this as well. It's worth mentioning that things may be a little different depending on which dichroics/beam splitters are in front, but the above are pretty good starting points. In regard to Marck Schlomchik's comments about Alexa 647, there's no question that the absorption spectra for this fluor are the closest match for HeNe excitation. However, if your goal is two red laser fluorescences, it's going to be pretty tough compensating 647 from 633 or 660. Richard Haugland correctly points out that A680 might be an option for a red combination with A647, since it has a pretty nice secondary peak around HeNe excitation wavelengths. In our case, however, we're looking to simplify things by generating conjugate pairs that can be used on all our instruments. The limitation here is our modified BD LSR (which by the way is a fantastic instrument, and I have no commercial interest in BD), which by virtue of the compact and extremely complex lightpaths won't allow us to use 647 and 680 together (at least not without ordering a number of custom filters and dichroics; maybe if BD or Omega or Chroma wants to help us out we can...). So for us, using two red fluors excited by a HeNe with detection compatibility on all our instruments, 633/660 works and works quite well. -- Howard T. Petrie, Ph.D. Head, Laboratory of Developmental Immunology Memorial Sloan-Kettering Cancer Center Box 341, 1275 York Avenue New York, NY 10021 phone (212)639-2149 fax (212)794-4019
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