In our lab we have had discussions whether we should represent our flow cyotmetry data as fold-change or background subtracted. We vaccinate animals with antigens and later re-stimulate the splenocytes in vitro with the antigen, and we look at cell populations using surface staining and cytokine responses by intracellular staining. All our results are percentages. We usually have +10 animals per group, so we take the median of all the %-values and we plot that in a graph and perform statistics.
Example:
#1 Media only: 0.2% IFN-y+
#1 antigen restim: 1.2% IFN-y+
Whether to represent the results as 1% IFN-y or 5 fold change?
Thanks in advance!
Hi Leroy,
I believe the most appropriate way to represent such kind of experiments is the background subtraction (and the most frequently used in publications...), for each animal after excluding dead cells by an amine-reactive dye viability marker. The most stringent you are in your threshold of positivity (eg at least 0.05% after subtraction, or higher!) the better...but with a minimum of events in your cytokine-positive gate (which implies acquiring high numbers of cells).
Hope my comment will help in your discussions,
Marcelo
Hi, normally we subtract the background that we get in the FMO control. But we show the values for cytokine secretion in both unstimulated (after subtracting FMO value) and stimulated conditions (after subtracting FMO value) in the graph.
Best,
anchana
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