Hi Maris, I wonder if there is something in the Hoescht staining protocol that is raising the APC background, independent of the actual Hoescht signal? You could substitute something benign (like saline) for the Hoescht, then carry out the complete staining protocol and see if that changes the APC background. Happy hunting! Joseph. At 03:12 11/1/2002, Maris Handley wrote: >Hi Everyone, >Happy New Year to All! > >I am at a loss to explain why cells that are stained with Hoechst33342 are >increasing >the background in my APC signal...HELP! We were looking at DNA using >HO33342 (excited by >UV, emission through a 450/20 filter) and APC (surface receptor, using >647nm excitation >and emission through a 670/20 filter). There was nothing out of the >ordinary visible >during the alignment (no laser noise increasing the background of either >signal, etc). >We set the APC negative control within the first decade, looked at the >APC+ control, and >saw a distinct positive population. When we put on a sample that was >stained with HO >(APC-), the signal in the APC channel went into the higher end of the >second decade. >At first, we were splitting the two signals with a 510LP, and after >witnessing the >shift in background, we switched to a 640LP...we still saw the same shift. >We compensated the HO out of the APC signal, and the experiment seemed to >work as >expected (read: the researcher was happy with his results), but does >anyone have any >idea what happened? I've been looking at every spectrum figure I can >find, and have >looked at the filter specs and I can't find any reason for that >shift. Any advice, >as always, is sincerely appreciated. Thanks, >Maris > >Maris A. Handley >Dana-Farber Cancer Institute >J415 >44 Binney St. >Boston, MA 02115 >(617)632-3139 >maris_handley@dfci.harvard.edu -- Joseph Webster, Flow Cytometry Facility Centenary Institute, Sydney, AUSTRALIA. Phone +61-2-9565-6110
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