Hello all, I am trying to determine the expression of embryonic stem cell markers such as OCT4, SOX2 and other transcription factors in mesenchymal stem cells using flow cytometer. I block the cells with Fc block and i have the FMO controls. Do i need Isotype controls as well?
In general, FMO's help in gating strategy, i.e., to draw a gate for the negative population. On the other hand, the isotypes help to show nonspecific binding under the assumption that both the isotype and the protein-specific antibody has similar non-specific binding. If your antibody has zero/negligible non- specific binding, it will closely reflect your FMO control. Isotypes are good if you are trying to assess small shifts. FMOs will have small shits in population related to compensation, which will mask any small changes. If you expect a big shift, you can get away with FMO’s, conservative gating and if possible a control or untreated sample.
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