You should have no problems at all using PI in a 3-color system; however, your other two colors should be FITC and PE. PI's emission is strongest in the FL3 channel; you can then compensate between FL3 and PE (FL2) to remove the contribution of PI into the PE channel. Note you will probably have to turn down the voltage on FL3 significantly, because PI is so bright (or, you could titer the PI to lower concentrations, but I wouldn't recommend this). Note that we frequently do 3-color + PI experiments--i.e., stain with FITC, PE, and Cy5PE, and PI as well. The PI (in dead cells) is so much brighter than nearly all Cy5PE stains that the dead cells can be easily excluded (and you don't have to worry about PI compensation, since you are excluding the PI+ cells). However, since you wish to enumerate PI+ cells, you need to devote one channel to its measurement--hence, use the FITC/PE/PI setup noted above. By the way, I recommend against 7-AAD; it's not as bright as PI. However, you might want to compare using it also in a FITC/PE/7-AAD set up. mr
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