I think is too difficult to explain over email with no figures to point to. Setting gates is not always straightforward. It can be a little bit subjective. Is there a resident flow expert at your institution that can "show" you how to do it? One thing I will say is that using quadrant analysis on a 2-parameter dot plot is not always feasible. You may have noticed that sometimes you get more than 4 populations. Even if you do only see 4, they are not necessarliy rectilinear enough to fit neatly into quadrants. Also, you don't mention how you are gating on the light scatter plot. I think you could really use some "hands on" help. David McFarland SmithKline Beecham Pharmaceuticals
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