>From my recollection of the XL system II software that I assume you are using the manufacturers recommendations are correct in that you will need as your 3rd histogram FS on Y and FL3 or FL4 on X, however you must adjust the voltage of FL3 or 4 depending on which you have used so that the bead population appears discreetly apart from the FS population on the Y axis , but perhaps in the 2nd or 3rd log decade on FL3 or 4. This will enable you to place the cal region around what should be a discreet bead population for your absolute counting requirements. It is also sometimes advisable to negatively gate out the beads from your histograms you are using for analysis by Boolean logic. Ian
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