I am trying to do absolute count for the first time using flowcount flouroshperes. I stain whole blood with anti-CD4-PE and anti-CD3-FITC antibodies for the analysis. I have a FS Vs SC histogram and a anti-CD3-FITC Vs. anti-CD4-PE histogram with all required compensations. For the third histogram where I am supposed to draw the "Cal" (counting) rectangular region, I have FS on the Y axis and what parameter I should put on the Y axis? The manufacture's insert says I should put FL-3 or FL-4 channel on the X-axis, but if I do that I do not get appropriate population in the "cal" region and the counts are off the range. Logically, it seems to me I should draw FS (on Y-axis) Vs. anti-CD-4-PE (FL2) on the x-axis with the "cal" region around the CD4+ population. What is the right way to do it? I hope many of you may be doing this as a routine, so I would really appreciate your valuable comments and help on this. Thanks Sathi
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