Dear Fellow Flowers, Since I have struggled with the issue of three color compensation myself (and I do it all the time) I just want to say one thing. Just when you think you have it all figured out you find out that you don't! The standard practice of compensating with single color tubes works most of the time but not always. Bunny has added the additional variable of permeabilization to the mix so she needs to compensate with permeabilized cells as well. Even when you think everything is going well you may have one combination of antibodies that don't like the compensation settings that work for everything else. I've seen the software for after-the-fact compensation and it looks like a great tool to have. I think the only limiting factor to this is that it won't help you if the sample has been overcompensated (if I'm wrong about this someone please correct me). The take home message from all of this (in my opinion) is that compensation must be determined empirically by skilled operators. Margaret > >Lest anyone draw the wrong conclusion, setting your compensation depends >very much on the brightness of the compensation standards! You can only >count on your compensation being correct for events as bright or dimmer than >the standards. Mario wrote the book on this, but his statement might be >misconstrued. Moral: If you use compensation standards at all (as opposed >to setting compensation for each individual pair of reagents) the standards >should be as bright as your brightest reagent. > >Happy Y2K. > >Albert > > > >Albert D. Donnenberg, Ph.D. >Interim Director >Bone Marrow Transplant Program, UPCI > >Phone: (412) 624-9596 >Fax: (412) 624-9624 > > > > >By the way, I want to remind everyone that compensation settings DO >NOT DEPEND on the brightness of a reagent! Therefore, you should >always use the brightest reagent you have in any given channel to set >compensation! >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Margaret M. Tropea Critical Care Medicine Department National Institutes of Health (ph) 301-496-7752 (fax) 301-480-3389 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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