>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). Interference filters (as opposed to glass absorbance filters) will tend to pass light again in part of the spectrum far removed from the bandpass. For flow they need to be built upon colored glass blanks to prevent this, instead of clear glass. That might be happening with your 670/20. If you have a spectrophotometer you could check it down at 450 nm. Otherwise, if you have an argon laser, set it to the 457 nm line and see if the filter blocks it. Marty > 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 -- Marty Bigos Director, Flow Core Gladstone Institute of Virology and Immunology Building 3 SFGH Rm 509 415-695-3832
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