If we are looking at a receptor expression on T cells (IL-10R) is it better to use the % of positive cells, mean fluorescence intensity, or median fluorescent intensity?
Just to add to Rezas answer: if the separation of positive and negative cells is crystal clear, % positive cells may be an adequate measure.
However, if you find it difficult to determine the exact limit between positive and negative cells, IL-10R MFI in your entire population is the most objective measure of IL-10R expression.
Of course, if you can also look at IL-10R MFI within your positive gate. This is only meaningful if the expression levels vary to a certain degree (e.g. for CD4 it would make little sense as CD4+ cells express uniformly high levels).
Both of the MFI or percentage of positive cells are important. The point is that which one of presence or abundance of the target (IL-10R) is meaningful for you. If the abundance of IL10R means something special, both of them must be addressed. But if not, just % IL-10R positive cells must be significant.
Just to add to Rezas answer: if the separation of positive and negative cells is crystal clear, % positive cells may be an adequate measure.
However, if you find it difficult to determine the exact limit between positive and negative cells, IL-10R MFI in your entire population is the most objective measure of IL-10R expression.
Of course, if you can also look at IL-10R MFI within your positive gate. This is only meaningful if the expression levels vary to a certain degree (e.g. for CD4 it would make little sense as CD4+ cells express uniformly high levels).
When I am reviewer, I always become suspisious when people show mean values, without the exemplary plots. If you use MFI show at least some exemplary histograms. Also, Define MFI, is it mean fluorescence intensity or index? I like the index better, because here you include the intensity of the respective isotype control into your data. If you are looking for small differences, it is hard to compare different experiments because minimal changes in the photomultipliere-settings of the FACS or in the staining quality gives your significant changes. So always do an isotype control....which you should do anyway :-)
Thanks. We always to isotype controls. However, I am not sure how to calculate the mean floroscence index. Will find out and see.
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