Hi- We have run these 3 dyes in our facility for quite a while. There are several technical issues involved. The first is to make sure that the two tandam conjugates have good energy transfer. This can vary from lot to lot. Second, for the Cy7PE especially, but for the others as well, you want to make sure that you have the optimum light collection setup. This means that a) you need red-sensitive pmt's (hamamatsu R3896 or equivalent) and b) optimal filters. With those 3 dyes a 575/25 bandpass for PE, 700/80 bandpass for Cy5PE, and a 750 long pass for Cy7PE are good choices. Also the beam splitters need to match the above filter set-up as well. The reason for all the above hulla-baloo is that when you are collecting out in the far red, the actual amount of light there is relatively low, much lower that for FITC, even though the separation between the unstained background and stained cells may be quite good. So although single stains with each reagent may look fine, when doing multiple stains, the compensation interactions and the inherant statistical variation in the low level light signals can cause problems (smearing of the backgrounds, mainly). Below is the spillover values for these three colors as well as FITC. Fluor PE Cy5PE Cy7PE Fluor 1.0000 0.3532 0.0712 0.0044 PE 0.0091 1.0000 0.2588 0.0150 Source Cy5PE 0.0004 0.0241 1.0000 0.0549 Cy7PE 0.0043 0.2138 0.0822 1.0000 For all the PE-based reagents, there are enough interactions between them so that full 3x3 compensation is usually necessary for obtaining interpretable results. The addition of FITC further complicates the interactions. In general, after compensation you will find that the the background on all three measurements will increase. Best of luck. >Dear All, > >does anyone have any experience in analyzing preparations stained with Ab's >conjugated with PE, PE-Cy5 and PE-Cy7 at the same time. Are there any >problems associated to this kind of stainings? > > >Carl-Magnus Högerkorp >Stem cell laboratory >Department of Internal Medicine >University Hospital of Lund >Sweden Marty Bigos Operations Manager Stanford Shared FACS Facility bigos@stanford.edu (voice) 650-723-6959 (fax) 650-725-8564
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