Linda, The ideal choice of fluorochromes for performing 4 color flow cytometry will depend on several factors including the type and configuration of the flow cytometer that you are using, the sensitivity required from the third color, and the availability of antibodies in the format of choice. All three of the reagents you mentioned (PE-Cy5, PE-Cy7, and PE-TR) can be used with FITC, PE, and APC. Each of these reagents has advantages and disadvantages vs the other options depending on your needs. I assume based on your question that you are probably using a BD FACSCalibur since both PerCP and APC were previously options for you. PE-Cy5, (TriColor from Caltag, Cychrome from PharMingen), has the issue of direct Cy5 excitation by HeNe and red diode lasers as you noted for Cychrome. This problem can be quite easily handled by compensating the signal out of FL4 (increase FL4-%FL3 compensation as appropriate). If you are using a machine that does not have the capability of performing cross beam compensation then the FL4-FL3 compensation must be performed by software during data analysis. PE-Cy5 also requires a small amount of compensation against the PE channel. The advantage of PE-Cy5 is that it's the most sensitive of the tandem dyes you mention as third color candidates and it is also the most widely available. PE-Cy7 is not significantly excited by red lasers and thus does not require compensation to correct for signal in the APC detector. PE-Cy7 does require significantly more compensation against the PE channel than does PE-Cy5, due to the less efficient energy transfer between PE and Cy7. This less efficient energy transfer, combined with issues of detecting the longer wavelength emissions from Cy7, make this reagent somewhat less sensitive than Cy5 although still quite usable with the selected antibodies it is conjugated to. PE-TR like PE-Cy7 does not have compensation issues with APC. It does require a significant amount of compensation against PE and is best suited for use on either Coulter instruments configured for running ECD or on larger cell sorters that are equipped with the proper optical elements. PE-TR can be used on a FACSCalibur, but the FL3 channel misses a portion of the PE-TR fluorescence and sensitivity is thus reduced on these instruments. I hope this answers your question. Best regards, John McChesney CALTAG Laboratories jmcchesney@caltag.com ---------- >From: linda duncan <opt059@abdn.ac.uk> >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >Subject: Advice on 4 colour flow >Date: Thu, Oct 18, 2001, 12:50 PM > > >Dear All, >For various reasons, I will be purchasing antibodies from >Caltag, and need to get a combination that will work. I >know that I can use FITC/PE/PerCP/APC but cannot use >FITC/PE/Cychrome/APC because of the overlap in excitation >between Cychrome and APC,but Caltag has three different >fluorochromes on FL3: PE-TR; PE-CY5; PE-CY7. Which FL3 >Fluorochrome from Caltag will work with FITC, PE,and APC? >More importantly which ones won't work. Please help!! > >---------------------- >Linda Duncan >Dept Ophthalmology >University of Aberdeen >Medical School >Foresterhill >Aberdeen >AB25 2ZD >l.duncan@abdn.ac.uk > > >
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