Under almost all conditions it's appropriate just to leave the FMO channel empty as a measure of the population's autofluorescence.

The indiscriminate use of isotype controls is inappropriate for a number of reasons - the most important of which is simply that every monoclonal Ab has individual background binding characteristics due to its unique antigen-binding site. Many companies even specifically select their "isotype controls" based on low binding(!)

Isotype controls can have a place if you are concerned about binding of the Fc region to Fc receptors. Otherwise there are better biological background staining controls, eg:

-is there another population in your sample with similar surface characteristics to your target population that you are certain would not express your marker?

-preincubation with a molar excess of unlabelled antibody of interest (isoclonic control)

-Can you get a knockout mouse/cell line?

Under almost all conditions it's appropriate just to leave the FMO channel empty as a measure of the population's autofluorescence.

The indiscriminate use of isotype controls is inappropriate for a number of reasons - the most important of which is simply that every monoclonal Ab has individual background binding characteristics due to its unique antigen-binding site. Many companies even specifically select their "isotype controls" based on low binding(!)

Isotype controls can have a place if you are concerned about binding of the Fc region to Fc receptors. Otherwise there are better biological background staining controls, eg:

-is there another population in your sample with similar surface characteristics to your target population that you are certain would not express your marker?

-preincubation with a molar excess of unlabelled antibody of interest (isoclonic control)

-Can you get a knockout mouse/cell line?

Dear Evan,

as Andew said already, you need to know what you want to control for. If you want a control for unspecific binding of an antibody to Fc receptors due to its Fc isotype, then you should also use an isotype control...

Isotype controls are obviously more important when you are dealing with cell types that do express a lot of Fc receptors. For your cell type (CD11b myeloid cells) I would very much recommend using one. For, let's say T cells, unspecific Fc receptor binding, may not be such an issue.

Isotype controls (as well as other measures, e.g excluding duplicates...) are also important if you are expecting a very small positive population, as only a small number of false positives will change your results drastically.

Yes, KO cells stained exactly the same way would be perfect, but that's unfortunately not always possible. If you do an initial trial on your cells, having one control with and one without isotype control antibody, you may be able to figure out if you need one or not. If you can't do any of that, my personal recommendation for your cell type would be to use one...

Thank you both for your solid answers. I want to evaluate myeloid cell "readiness" to exit the bone marrow and fight an infection, comparing a treated and untreated group. I will try both FMO approaches (isotype, empty channel) to see if there are major differences in measuring the amount of CxCR2 expression.

Hi. Note that FMO is not the same as using isotype controls. If you have a four colour experiment and want to run FMO controls you need 4 tubes: each one will have all the antibodies except one. This will allow you to see how your antibodies (when together) spill over that particular channel. In isotype control you usually can use all your isotypic controls in the same tube... So, different things. Fluorecence Minus One... that is what FMO means. Another thing is a simple autofluorecence control where you simply leave those channels empty... and measure de autofluorescence of your cells. The FMO are usually employed when designing antibody pannels. Isotype controls or empty channels are used in a experiment basis.... Hope this help

We use FMO totally w/o fluorochrom. It only\ shows a spillover to the channel and helps you with gating. Isotype controls you can use in parallel, nevertheless we prefer to use FC blocks instead (see Cytometry 1999;38:280). You can read more on http://flowjo.typepad.com/the_daily_dongle/2011/09/fmo-vs-isotype-controls.html

Ludek,

I'm not sure I got the reference right. Do you refer to "Increased peripheral blood gamma delta T-cells in patients with lymphoid neoplasia: A diagnostic dilemma in flow cytometry." by McClanahan et al.?

Hi Enrique, full citation is Keeney M, Gratama JW, Chin-Yee IH, Sutherland DR. Isotype controls in the analysis of lymphocytes and CD34+ stem and progenitor cells by flow cytometry--time to let go! Cytometry 1999;38(2):280-283.

Thanks a lot!

Hi! I also have a question regarding FMO controls.

I want to use a 6-color panel to check for some surface markers in NK cells. I would like to use FMO controls for my samples, but I would like to measure the expression of these 6 molecules in samples with different treatments. Should I use FMO controls for each treatment, of maybe using the FMO with the control treatments would be also accurate??

I forgot to mention that my problem is that I have to measure all the different treatments on the same day, that´s why I would like to know if it is possible to avoid using so many FMO controls for my stainings...

For each particular combination of fluorochromes (icorresponding to a certain panel), some fluorochromes' spill-over on another will be so great that compensation is not enought to correcto for it., and you have to use an FMO. However, there are cases in which compensation is enough, and therefore you can ommit all the fluorescences (fluorocrhomes) in your FM control for which the spill over is corrected sufficiently with compensation. You can determine this for your particular pannel by plotting fluorescence detected in PMT X versus fluorescence of another fluorochrome Y in Y^negative and Y^positive particles. You need to compare the spill over before and after compensating. There you can see if compensation corrects all false positive X fluorescence. For instance, FITC receives spill-over from other fluorocrhomes that may be corrected with compensation, and thus you spare work and money by ommiting a fluorescnece minus FITC. There can be cases in which you need a, say, fluorescence minus3 or minus 2. It depends on the particular experiment.

One additional point. FMO is an optical control that corrects for false + fluorescence stemming from spill over. It does NOT correct for unspecific binding of antibodies. As mentioned by Ludec Sefc, you control that by blocking Fc or using isotype controls. Many times you don't need to block unspecific binding because it is minimal. Again, this depends on the particular clones you are using.

When I have many samples - like you do, I make a mix of all the samples and aliquot for FMO's and single stains from that pool of cells. So just one set of FMO's and cells for all these controls.

Also, I primarily use FMO's to help to set gates. They also can give a clue about whether compensation is off, but I find that happens rarely.

Thank you very much Enrique and Felicity for your advices! I have compensation settings for all the fluorochromes that I want to use in my samples, so I will try to measure without FMOs and then, if I see some spill overs within some fluorochromes then I will use FMOs controls just for them, so then I will save time and money as you have mentioned Enrique : )

Hitherto I was also making a mix of all the samples and use the pool for FMOs, but I was not totally sure about if that was correct.

Hi,

I would be careful about mixing very different samples. Let's say that you have control cells (non-activated) and activated cells - they might completely different properties. I tend to do FMO staining tube (without isotype Ab) for each condition in the experiment (one representative sample of the group).