I would like to know if using FMO controls is better than using isotype controls for flow cytometric cytoplasmic staining eg. MPO-PE with TdT-FITC. What is the best protocol for using FMO controls?
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Hi Jan,
I assume you are using a blue 488nm laser for both FITC and PE?
Isotype controls and FMOs achieve different things, which are especially important in intracellular staining.
ISOTYPES
I agree with Alka that isotype controls do not provide a perfect control for non-specific staining. However, in intracellular flow cytometry the are more necessary -- whilst it is not a perfect approximation of non-specific staining, the presence or absence of signal after intracellular staining gives you a good read-out for whether you have washed your cells well enough. Antibodies can stick and be trapped INSIDE the cell with insufficient washing more easily that just on the surface.
It is worth mentioning that if you publish a figure for intracellular staining, the reviewer will ask for an isotype control.
I would recommend using an isotype control for intracellular staining.
FMOs
FMOs are a different story. An FMO (fluorescence minus one) is design to show the level of background that is present in a particular fluorescent detector. It is designed to be used as a gating control, as the increase in background from fluorescence spillover is not shown using isotype controls. This is useful primarily for markers that have a RANGE of expression, such as chemokine receptors, which will literally have signal all the way from none, to a little, to a lot.
When different fluorescent signals are compensated, this can result in "spreading error", especially in far-red detectors. Essentially the compensated population (in this case FITC) signal spreads out in the other detector (in this case PE). This results in an increase in the negative/background in the PE detector. However, there is not very much spreading error from FITC into PE. If you are using a blue 488nm laser for FITC and yellow/green 561nm laser for PE, then you won't have any real spillover between the two.
To make an FMO, it is mixed up the exact same way as your antibody mix, MINUS the specified antibody. In your case -- the FITC FMO has only PE in it, and the PE FMO has only FITC in it.
If your markers have a range of expression, or have very low expression, then I would include an FMO. There is also a chance a reviewer will ask you for this as well.
Hope this helps.
Yes ! The usefulness of isotype controls is heavily debated, since a number of years already, and consensus is that they are absolutely not useful in establishing non-specific antibody binding. An FMO-control (Fluorescence Minus One) is a control sample containing all of your antibody labels, except one. Theoretically, you'd need to prepare an FMO-control for each of your antibody labels. They will allow you to correctly establish spillover-related contribution to background in each of your channels.
HTH
Hi Jan,
I assume you are using a blue 488nm laser for both FITC and PE?
Isotype controls and FMOs achieve different things, which are especially important in intracellular staining.
ISOTYPES
I agree with Alka that isotype controls do not provide a perfect control for non-specific staining. However, in intracellular flow cytometry the are more necessary -- whilst it is not a perfect approximation of non-specific staining, the presence or absence of signal after intracellular staining gives you a good read-out for whether you have washed your cells well enough. Antibodies can stick and be trapped INSIDE the cell with insufficient washing more easily that just on the surface.
It is worth mentioning that if you publish a figure for intracellular staining, the reviewer will ask for an isotype control.
I would recommend using an isotype control for intracellular staining.
FMOs
FMOs are a different story. An FMO (fluorescence minus one) is design to show the level of background that is present in a particular fluorescent detector. It is designed to be used as a gating control, as the increase in background from fluorescence spillover is not shown using isotype controls. This is useful primarily for markers that have a RANGE of expression, such as chemokine receptors, which will literally have signal all the way from none, to a little, to a lot.
When different fluorescent signals are compensated, this can result in "spreading error", especially in far-red detectors. Essentially the compensated population (in this case FITC) signal spreads out in the other detector (in this case PE). This results in an increase in the negative/background in the PE detector. However, there is not very much spreading error from FITC into PE. If you are using a blue 488nm laser for FITC and yellow/green 561nm laser for PE, then you won't have any real spillover between the two.
To make an FMO, it is mixed up the exact same way as your antibody mix, MINUS the specified antibody. In your case -- the FITC FMO has only PE in it, and the PE FMO has only FITC in it.
If your markers have a range of expression, or have very low expression, then I would include an FMO. There is also a chance a reviewer will ask you for this as well.
Hope this helps.
Thank you Thomas, your explanation on Isotype Controls and FMOs was very useful. I appreciate your response, it has made me understand this concept much more clearly.
Thomas,
I do agree to the fact that the situation for intracellular staining is really delicate.
The problem with isotope controls is that they still are a different conjugate from the conjugate you're trying to establish non-specific staining for. This means that there is no guarantee that the actual fluorochrome has the same emission spectra. This becomes especially obvious for dual-conjugates (e.g., PECy5; think of conjugate differential degradation because of light-exposure). Then there is also the possibility of F/P ratios being different. Then there is the issue of titration of the antibody … We all accept that that is crucial, but how do you titrate an isotope control ?! There is literature describing extremely different non-specific staining characteristics of "identical" isotope controls (from different providers). What to do with that situation ?! I often have the impression that isotype-based estimation of non-specific binding is somewhat too arbitrary.
Finally, no, we excite our FITC and PE from separate lasers, like you do.
Jan
You are right, I am using the CANTO II from BD with 3 lasers - blue, red and violet, with a blue 488nm laser for both FITC and PE, but I am also using 2 additional cytoplasmic antibodies CD79a PerCP-Cy5.5 (blue laser) and CD22 APC (red laser)with CD45 V500 (violet laser) for surface in the same tube. I have been using the other tube with isotype controls for each of the 4 antibodies. So in this case I will have to make 4 FMO tubes along with my cytoplasmic antibody tube.
What they are saying is all very correct and helpful. I would just like to add that for intracellular stainings for dim markers like cytokines I would suggest using the FMO, but do not leave one channel free, but add the isotype control. There is no need to do that for all the markers. If you stain for example for cytokine expression in CD4 cells, you would do the FMO-Isotype control for the cytokines, but not for CD4, CD8 or CD3. So the actual number of samples might be not so high.
Hello Thomas Myles Ashhurst ,
Your answer is very helpful to me, thank you so much!
Besides this, I want to ask you some other questions. Because these days I am stuck in the intracellular staining of IL-17 in PBMC. I tried to use different protocols, but in my experiment I always found the expression of isotype was higher than that of IL-17 as well as auto-fluorescent. If I took auto-fluorescent as a negative control, I could find lower expression of IL-17. The isotype I used is recommended by the company for anti-IL-17A. The intracellular staining is really so complicated. Do you have any suggestions?
Thank you so much!
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