The emmision from Alexa 350 peaks at about 440nm thus you should be using a 450BP25 or maybe a 450BP40 filter to capture light. Of course if you are running 488 and 350 colinear you must filter out the 488nm light. I have seen very good signals from this dye running a UV laser at 50-100mw. You should be able to get at least a decade of separation from negative to positive with CD4 antibody. I recommend using about 1ug of Alexa 350 secondary for 1-2x10E6 cells. Also be aware that you have to peak your signal with UV beads or equivalent at the detector to capture optimal fluorescence. Good luck. David M. Dombkowski dombkowski@helix.mgh.harvard.edu Flow Cytometry-Pathology-CNY rm7017 149 13th Street Massachusetts General Hospital-East Boston, MA 02129 Tel. (617)726-1683 Fax.(617)724-3164
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