Flower's: One of my users has an interesting problem, and I'm hoping that someone can help educate me why it might be occuring. We are using an LSR-II and have pmt configuration of 525LP in front of a 530/30 BP for YFP and 635LP in front of a 670/14 BP for 7AAD. The cells are eYFP inducible, and we are using 7AAD as a vital dye. The compensation was set with single stains using the AUTOCOMP feature of DIVA software. Everything is fine, except for cells that have an extremely high level of YFP expression. In these cells, there is no clear 7AAD positive fraction, and about 20% of the YFP bright cells are being lost based on the gates set off of the negative (uninduced) cells. However, by nudging the compensation up just a bit (from 8.27 to 9.27), the plots look normal again. Attached is a pdf showing the differences. Has anyone else had this problem? Why might this be happening? Thanks Tim Timothy Bushnell, Ph.D. University of Rochester Center for Human Genetics and Molecular Pediatric Diseases Flow Cytometry Facility 1-11314 585/273-1361 <<YFP-7AAD.pdf>> This attachment - 'YFP-7AAD.pdf' - 324.01 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/b5157a6f61deafb41314942846bbe4113ed87bdd.pdfReceived on Wed Mar 24 14:38:00 2004
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