I would like to determine which subset of cells have taken up an antisense oligonucleotide, and to what extent. Does anyone have a good method for this? I have tried cotransfecting my cells with a FITC labeled irrelevant oligo (a preliminary study before getting the actual antisense oligo labeled), and I got what looks like a lot of non-specific labeling of the cells. I assume this is due to sticking of the fluorescent oligo on the surface of the cells. The dead cells took up even more of the fluorescent oligo. Basically, the fluorescent oligo was a good label of dead cells. In fact, when I labeled the cells with ethidium monoazide and fixed them, I got an interesting result (at least to me). In the control cells without the FITC oligo, the dead cells stained with EMA were moderately fluorescent in the FL2 channel. The same dead cells with the FITC oligo were very negative for FL2, but had the bright FL1 staining from the FITC. Could there be some quenching going on? If so, I was wondering whether it might be possible to use this quenching phenomenon to identify live cells that had taken up the oligo. For instance, could you have the cells constitutively expressing a fluorescent marker, and then transfect them with the fluorescent oligo and see which cells quenched? H. Elizabeth Broome, M.D. Department of Pathology University of California, San Diego 9500 Gilman Drive #0612 La Jolla, CA 92093 ebroome@ucsd.edu Phone (619) 534-5216 FAX (619) 534-7415
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