I treated cells with certain kinase inhibitors (including Vehicle) and measured phosphorylation levels (single stains with specific primary ab and AlexaFluor488) with FACS. I analyzed my data in FlowJo (first gated for debris, then excluded doublets). I excluded the background signal, and created histograms based on the marker for FL-/FL+.
I am not sure whether I should interpret my data as Fl+ percentage or should I use statistical data (for the single cells gating) such as Mode, Median or Mean? and if so, which one?
I tried both ways, and I get differences in phosphorylation levels for some of the antibodies. That's why I hesitate which one to use. Thanks!
Hey!
I would use the Mean Fluorescence Intensity (MFI) and then normalize it to your control.
Klara Terörde and Martin Böttcher , did not manage to thank you for your help!
Hi Margarita,
it´s right - it´s not very meaningful to show Phospho-flow data as % positive... Just an addition to Martin´s hints: It´s better to interprete data as "delta MFI", i.e. MFI of sample X stained with the phospho-specific mAB
MINUS
MFI of sample X stained with an appropriate Isotype control (i.e. Ig x of the same host with irrelevant AG-speciificity coupled to the same fluorochrome)
This is the most reliable interpretation since some inhibitors/vehicles/compounds added for stimulation change the " intrinsice fluorescence" of cells (i.e. "background"). If possible (enough cells), I divide each sample in half after stimulation: one part stained with Isotype control and one part stained with the P-specific mAb...
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