Bunny, you have just realized the difference between the configuration of the LSR vs the Facscan but all is not lost. You are right in that the 3-part dif of SSC vs FSC is not as elegant as the Facscan but I have been able to get very high quality diff's by playing with the adjustment of both FALS and SSC gains. It may require a little adjustment of the SSC dichroic to maximize the separation in SSC. Beware when adjusting this dichroic that you may notice a change in FITC vs PE or in the UV signal. There is a "synergy" between the dichroic angles and maximum signal to PMT that is essential to acheive. You may also want to play with the angle of PMT to acheive maximum signal. The compensation between the FL3 and APC is not able to be done.The B.D. software will not cross beam compensate so that you will have to consider software compensation using WinList or FlowJo which is really quite easy to do. Try WinList using the compensation toolbox and saving file with newly compensated parameters. My observation is that you can have some effect on this compensation problem when adjusting say FL3-FL1 or by adjusting the gain of PMT. You need to spend some time playing with the optimization of this instrument in order to acheive an understanding of how everything works. David 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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