At 9:04 AM -0500 2/9/01, Timothy Singleton, M.D. wrote: >You could use 7AAD to gate on live cells for analysis. But why? PI is much brighter than 7AAD and therefore you can use it in combination with cell surface markers on the same channel. 7AAD requires that you devote a detector to it. This means on a 3-color machine your doing only two-color immunofluorescence... with PI, you are still doing 3-color IFA. Furthermore, with 7AAD you must properly compensate against 7AAD fluorescence (see <http://nucleus.immunol.washington.edu/Research_facilities/Apps/7aad.h tml>) otherwise you will not properly include live cells. PI is so bright that compensation usually isn't an issue. As long as you properly compensate your regular fluorophores, it is relatively trivial to pick out the dead cells (by PI, and ForSc often helps). Once you gate them out, there is no problem with analysis in any channel. Finally, the protocol with PI seems so easy--you only need to incubate 5 minutes... we typically include PI with our final wash medium or our final resuspension medium--in the end, there is no separate incubation. mr (PS, Yes, I know there are some 7-AAD fanatics out there, who will take issue with my response... Before we raise a tempest in a teapot, let's just agree that the most important thing, whatever dye you use, is to do some form of live/dead discrimination beyond just Forward scatter gating).
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