We've just started running these fluorochromes, FITC/PE/PE-Cy5 (PC5) or PerCP or PerCP-Cy5.5/PE-Cy7 (PC7), with the Coulter's new filter kit and with Coulter's Advanced Digital Compensation (ADC). Coulter's kit has the 675(?) band pass filter for the third channel, but we are now testing a 650 long pass for better sensitivity. The band pass filter is okay, but we get a stronger signal for PerCP-Cy5.5 with the long pass. The main advantage is that PC5 crosstalks into the PE channel less than PE-Texas Red (ECD). There are compensation issues. Different antibodies have slightly different optimal compensations with the tandem dyes. However, I think that we will be able to overcome this obstacle for routine clinical cases. We are adjusting the color compensation with single colors individually before mixing these together. Post-acquisition software color compensation is the ideal way to go, if it's available, although it might be time-consuming. This is not available with Coulter's Expo 32 analysis software for the XL but is available for Coulter's new five color instrument. If compensation is done during acquisition, some antibodies would likely be somewhat over- or under compensated. You have to test each antibody to make sure that it works. For example, we found that CD19 PC7 was too weak in our hands. Tim Singleton, MD Flow Cytometry William Beaumont Hospital Royal Oak, MI >>> Carol Oxford <cloxford@UCDAVIS.EDU> 05/23/02 01:59PM >>> I have an investigator who is interested in running four colors on our XL, but instead of FITC/PE/PETxRed/PECy5, he wants to use FITC/PE/PECy5/PECy7. Has anyone tried this? I have some ideas based on what we do on our MoFlo- I think it's just a matter of a new dichroic and band pass, but I wanted to see if there was some advice from someone who has it working. Thanks! Carol Oxford --------------------------------------------------------------------------- Carol Oxford _| _| _|_|_|_| _|_|_| Manager, UC Davis Optical Biology Lab _| _| _| _| _| Tupper Hall, rm 3425 _| _| _| _| _| University of California _| _| _| _| _| Davis, California 95616 _| _| _| _| _| (530) 752-7205 _| _| _| _| _| (530) 752-4548 fax _|_|_|_| _|_|_|_| _|_|_| (530) 759-4457 pager cloxford@ucdavis.edu University of California, Davis ---------------------------------------------------------------------------
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