Hi Paula If I understand right, the numbers would be different because in one case, the percentages on the dot plot are based only on the scatter gate, so it would be the equivalent of saying "out of all lymphocytes, x% of cells are positive for both an activation marker and a "FITC" marker. However, the numbers on the histogram would (or should) represent the percentage of the scatter gate AND the PE gate, and therefore be the equivalent of saying " x% of cells out of all activated cells is positive for "FITC" ". These should be the same as adding up PE+ and PE+FITC+, then getting what percentage the PE+FITC+ are of this sum. (assuming the markers and quadrants are the same). I think you could use either the dot plot or the histograms, depending on what differences you are trying to show or how big these differences are (generally doing the gate to histogram, or percent of total PE+ should give bigger numbers and therefore bigger differences) Artur PS. I may be wrong on all this, in which case it is only a suggestion :) ********************* Artur Plett, PhD IUPUI - School of Medicine-- Hematology/ Oncology 1044 W. Walnut St. Indianapolis, IN 46280 pplett@iupui.edu (317)274-0352 > -----Original Message----- > From: Paula Lavery [SMTP:plaver@po-box.mcgill.ca] > Sent: Monday, December 06, 1999 13:04 > To: Cytometry Mailing List > Subject: Reporting results of 2-colour stains > > > Hello Everyone out in Flow-Land > > Back to Flow Cytometry 101... > > I've been having a debate with our grad student on the appropriate way of > reporting results of 2-colour staining. He is staining activated T cells > with a PE-conjugated activation marker and another FITC-conjugated > monoclonal. He wants to make a statement on Activated vs. Non-activated > cells. He prints up contour graphs with both colours and calls the UR > quadrant his "FITC+ on activated cells" and the LR quadrant his "FITC+ on > non-activated cells". I think he should be gating on activated cells (with > FL2 histogram) and then produce an FL1 histogram to get the "FITC+ on > activated cells". (Similar with non-activated cells, of course.) The > numbers > are substantially different depending on the method you use. Help please! > > Paula Lavery > Transplant Immunology Lab. > Royal Victoria Hospital > McGill University > Montreal (QC) Canada > plaver@po-box.mcgill.ca > > "If we knew what we were doing it wouldn't be called research, would it?" > -- Albert Einstein >
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