Sharon Bader wrote: > > I find that when I run an isotype control and then the labeled sample, the > negative population within the labeled sample is lower than the isotype > control. This results in my background marker to be higher than it should > be. Sometimes even cutting into my positive signal giving a lower percentage > positive population. Should I set my gate on the isotype control? or on the > negative population of my labeled sample? The use of isotype controls is based on certain assumptoions which don't always hold. I t assumes the fluorescence of your isotype population will match the fluorescence of the negatives in your test sample. This means the amount of nonspecific binding of the isotype antibody will be the same as the amount of nonspecific "sticking" of your test antibody to the neggatives. As you have encountered, often this is not the case. When your positive population is a distinct cluster which is well resolved from thenegatives, it is generally accepted that it is OK to move your cursor to split the 2 populations. For antigens like CD4 and CD8, positive s typically resolve into distinct clusters. For antigens like CD95 and HLADR, there is more of a continuous distribution and much more effort is required to obtain a value for percent positive. Regards, Tom ***************************************************************************** Thomas W. Mc Closkey, Ph. D., Director of Flow Cytometry North Shore University Hospital, New York University School of Medicine Boas Marks Biomedical Research Center, 350 Community Drive Manhasset, Long Island, New York 11030 ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866 *****************************************************************************
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