Maris What instrument are you using? HO33342 does emit significantly at that wavelength so you can get cross-talk between lasers. After I know what instrument you are using I may be able to suggest something. Larry At 11:12 AM 1/10/2002, you 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 Larry W. Arnold, Ph.D. Associate Professor Director, Flow Cytometry Facility Department of Microbiology and Immunology Lineberger Comprehensive Cancer Center CB# 7290 University of North Carolina at Chapel Hill Chapel Hill, NC 27599 Phone: 919-966-1530 FAX: 919-962-8103
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