This is rectifiable by changing your display settings (some more background here: ). So just because your live-dead looks bad, does not mean your FITC or PE will. This will happen on a channel by channel basis. But sometimes, for kind of complicated reasons but usually sample-related, the baseline restore process (a subtraction process that DiVA instruments do on a event-by-event basis) will give a larger negative variance than usual. You are likely using a default Flowjo width basis of -10, which is fine for most fluorescence channels most of the time. The reason the events look fine on DiVA but not FlowJo is because DivA adjusts the display settings to accommodate changes in the negative fluorescence (), while in Flowjo this needs to be changed by the user. The issue you have is related to baseline restore and the display settings on your Flowjo. However, this usually requires dropping both the FSC voltage and the threshold setting during acquisition. It is possible to simultaneously visualise bothВ small and large cells on the FSC axis if you display the data in log form. If youвЂ™re only interested in the leukocytes then this isnвЂ™t really a problem per se, and simply reflects the limitation of the machine to display such a large range of cell sizes on a linear scale.

Without knowing these factors I would predict that your FSC is slightly too big and the stomal cells are causing the вЂњproblemвЂќ. What was the ratio of leukocytes to stromal cells? (if 1:100 then this is obviously predictable/acceptable, but if it is, say, 1:10 then itвЂ™s possible that you could be losing blasting leukocytes off the axis).ДЄlso, are you looking at the data on a log or linear scale? This is the FSC vs SSC plot, correct? Not FSC vs a fluorescence parameter? The cells can only be вЂњtoo bigвЂќ on FSC and SSC, but they can be too small on fluorescence channels due to compensation or baseline restore.Дid you use a BD machine when running the samples? (my experience and comments are applicable to BD/DiVA, not necessarilyВ other machines).

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