>Andy, > >I've used Cy7APC for nearly 7 years now, and have never seen it fall >apart as you describe. Actually, what you describe sounds most like >someone put propidium iodide in the sample--is that a possibility? > >You should be able to tell if it is APC-only signal by looking at the >contribution of the "APC only" cells in the Cy7APC channel--and >compare it to a truly APC-stained sample. Only if the spillover into >the Cy7APC channel is exactly the same for a true APC dye should we >consider the possibility that the Cy7APC is falling apart. > >If it is falling apart, then you might want to check your >solutions--maybe you have something in them that causes it to break >down (however, we have used many types of fixatives without problems). > >Can you EMail me some of your datafiles? Send me a collection that >has unstained cells, APC-only, and Cy7APC-only stained cells (i.e., >your comp samples)--before & after compensation, if possible! > >mr Mario, Thanks for your input here. I think PI is unlikely in that as far as I'm aware PI is poorly exited by a HeNe (at least in all my data charts for it). However I have seen a similar result for the Red670 dyes. I don't have the data files. I did see the exact same intensity at the same position on a plot for PharRed in the APC channel as the APC dye alone. So I concluded that the batches we were using were breaking down. I haven't used PharRed since, but am willing to it try again. It maybe that I have been unlucky in the batches that I have used. NB I have only used 2 different batches and make no claim about the integrity of the dye as a whole. I would like to investigate this problem further and thank everyone for their input. I have not heard of anyone else having this problem so it maybe that we are doing something (as Mario suggested), so I would like to investigate this further. If anyone else has any advice please let me know. Thanks Andy. Andy Riddell PNAC Division MRC LMB Hills Road Cambridge UK tel: (0)1223 402218 fax: (0)1223 412178 email ar3@mrc-lmb.cam.ac.uk
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