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
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