Compensation between two measurement channels, say FITC and PE, is determined by how much one day, say FITC, is detected (or spills over) by the PE detector. With more than one laser, there is an additional effect. A particular dye may be excited by both lasers and independently detected by measurement channels for each laser. This is not quite the same as "spectral spillover" mentioned above, but from the user point of view is treated the same using the various mechanisms for compensation, For PE-Cy5, the Cy5 component of this tandem energy transfer dye spectrally looks a lot like APC. It is efficiently excited by the red diode laser and has strong emission in the APC measurement channel. Its amplitude may be such that the subtraction electronics in the Calibur may not have enough gain to handle it. A suitable workaround would be to use analysis software and appropriate controls to do this. On our non-digital Vantage there is measurement overlap between the dyes PE-Cy7 and APC-Cy7 (we use a 633 nm HeNe) on that instrument). So for six color work we commonly use the analog circuitry to compensate what we can, and deal with these two reagents in the analysis software. Marty >Hi ya'll, > >Has anyone successfully compensated PE-Cy5 (FL3) and APC (FL4) on a >FACSCalibur? I have tried both during acquistion and post-aquisition >and have not had much luck. I am currently not recommending it, but I >am curious as to why it doesn't seem to work. > >Thanks, > >Julie > >Julie G. Nelson >Research Coordinator >Flow Cytometry Facility >Center for Tropical and Emerging Global Diseases >University of Georgia -- Marty Bigos Director, Flow Core Gladstone Institute of Virology and Immunology Building 3 SFGH Rm 509 415-695-3832
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