Resolution of dim fluorescence
Alan Salkind (nepsy@fiona.umsmed.edu)
Tue, 21 Mar 95 14:01:48 -0600
I am trying to analyze histograms which depict the expression of an
intracellular antigen in lymphocytes. Although there is a demarcation
between the overlaid histograms of the positive cells and the negative
control, there is considerable overlap between the two populations, making
analysis difficult. I tried subtracting the negative control histogram from
the "positive" histogram using the histogram tools application in Cell
Quest. I then divided (again using the histogram tools application) the
number of positive cells generated from this "new" histogram by the total
number of cells in the positive sample. I found this manipulation produced
misleading results. Is it reasonable to place a marker at the intersection
of the positive and negative histograms and consider events to the right of
the marker as positive? I am concerned that this may indicate more positive
cells than there really are. On the other hand, setting the marker based on
1 - 2% background fluorescence excludes the dim staining cells. I know this
issue has been discussed before on this BB, but I would welcome any new (or
old) thoughts on the subject.
Thank you.
............................................................................
.... Alan R. Salkind, M.D.
Division of Infectious Diseases, University of Mississippi Medical Center
E-Mail: nepsy@fiona.umsmed.edu
Tel: 601-984-5560
Fax: 601-984-5565
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