I am currently labeling cells for intracellular cytokines. I would like to know how valid using MFI is as a measure of the level of expression. My concern is the number of variables that could affect fluorescent intensity and the assumption that this level of intensity corresponds to antigen expression. Variables that include; 1. how fixing and permeabilizing affect fluorescent intensity 2. how the time between fixing and permeabilizing and reading it on the flow cytometer affects fluorescence intensity 3. exactly how many antibodies are bound to the cytokine 4. exactly how many fluorochromes are on the antibody Also, if you look at subsets within your population (ie; CD3+'ve cells) how can you compensate for this variable with respect to your population and cytokine expression. It seems to me that percentage positive is a more accurate measure of cytokine expression, since it is more QUALITATIVE, and MFI gives a falsely QUANTITATIVE measure of cytokine expression. I'm trying to understand this whole MFI concept. If anyone has any references that can explain its valid uses, that would be most helpful to me. Thank You Sharon Bader Children's Research Center Vancouver, B.C. Canada
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