In the flow cytometry experiment I will perform soon, I will use the unstained control to detect autofluorescence, the single stain control as a compensation control to determine the degree of fluorochrome spectral overlap, and the isotype control for non-specific binding. Since the device I will use is BD FACSCalibur and it is limited to only 3 colors and the number of samples is high, the number of tubes I need to prepare increases. Therefore, would I be wrong if I prepared FMO controls for only one sample and not all samples?
If you use FMO for your decisive marker, it would work. But technically, suggested by the application scientists of Flowcytometry manufacturers suggest you to run FMO for all the markers.
But it completely depends on your personal experimental set up. Run your experiments with FMO for only decisive marker few times and for all the markers, you will be able to see the difference in both. If you find no difference than Go ahead with Single control FMO.
All the best.
Below article will help you to understand FMO better.
Hulspas R, O'Gorman MRG, Wood BL, Gratama JW, Sutherland DR. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry Part B 2009; 00B: 000–000.
Also take a look at below:
https://bitesizebio.com/21127/all-for-one-and-one-for-all-fluorescence-minus-one-controls/
Best wishes,
No, it is not necessary to prepare FMOs for every sample, unless they contain different cell types.
For single stained controls (SSC), it is important that, if you use stained cells, the sample (or mix of samples) contains a clear positive population for that specific marker/stain, AND a negative population, which ideally should be represented by the same cell type as the stained cells (ie. it should have the same autofluorescence/aspefic binding). The positive population in the SSC should be as bright as the brightest sample you will analyze, otherwise the compensation you will calculate may be off for the brightest stained cells. You can use beads (ie. BD Comp Beads) which can be stained with you antibody of choice to prepare SSC (except for markers which will only work on cells, ie. PI, 7AAD, Hoechst, and other live/dead stains for example). You need one SSC for every fluorochrome you will use.
FMOs will assess the loss of sensitivity for a particular marker/channel due to spread deriving from fluorescences in different channels (ie. loss of sensitivity in the PECy5 channel due to PE and/or XCPCy5.5 emission). Therefore, they will be the ideal control to set the gate for that specific marker on cells stained for all the other markers (ie. set the gate for neg/pos cells in the PE channel using the FMO lacking PE staining). You will notice a shift in the threshold for positivity in the FMOs compared to the unstained control due to the signal from other fluorescences (even when properly compensated). You can, in some instances (ie. for cells/markers with high aspecific binding/staining), combine the FMO for a particular marker with the isotype control of the same fluorochrome. In this way, you can discriminate real positive from aspecific binding of the Ig molecule or the fluorochrome. Bear in mind that in some cases, the very dim signal from low expression markers can be less intense than the one produced by the isotype control Ab (when the aspecific binding of the IC is higher than the "real" dim signal of the targeted antigen). In any case, as a general rule, I would prepare one FMO for each fluorochrome/channel used, given that the cell mixture used contains cells positive for all markers (ie. I would you a representative mix of your samples). If you are analyzing different tissues, like bone marrow, spleen, peripheral blood, I would prepare a different FMO set for each tisse/cell type. A set of singe isotype control stained cells can provide information on the aspecific binding of your cells/Ab/fluorochrome combination. If there is relevant aspecific binding of your IC Ab, a second set of FMOs can be prepared with the IC for the same marker (ie. isotype control for PE on FMO for PE) to set the gate for positive cells.
If you have many samples, you should set up your panel (even if it is very simple) on a few, and then run all of them once you are ready. You can use special beads (ie. Rainbow calibration particles) to standardize your panel if you need to run it more than once.
If you use FMO for your decisive marker, it would work. But technically, suggested by the application scientists of Flowcytometry manufacturers suggest you to run FMO for all the markers.
But it completely depends on your personal experimental set up. Run your experiments with FMO for only decisive marker few times and for all the markers, you will be able to see the difference in both. If you find no difference than Go ahead with Single control FMO.
All the best.
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