Actually, isotype controls are not a particularly good control. They are rarely matched: the F/P ratio is not the same, and how do you know if you are using them at exactly the same concentration as the reagent of choice? If you don't know that the F/P ratio is exactly the same, and if you don't know if you are using it at exactly the same concentration as your antibody reagent, then it isn't the right control. Indeed, since each one of your commercial reagents is titrated by the manufacturer to give optimal signal to background, each one is sold at a different concentration of antibody. Have you contacted the manufacturers to determine the bottled concentration of each reagent you use, so that you can use the appropriate concentration of your isotype control? And then use a different isotype concentration as the control for each reagent in your various panels? If the answer to either of these questions is "no", then how can you assert that your isotype control actually gives you the correct amount of background binding in your experiment? i.e., your "isotype control" does no more than let you that there may actually be some background binding, but doesn't give you the ability to estimate how much. In fact, I've known people to "titrate" their isotype controls to get background binding that is less than what they think their positive should be. Hmm. In another way, isotype controls are rarely used properly: most people do a single sample that has all isotype controls in all channels. This doesn't help! One must use a control for which cells are stained with all reagents EXCEPT the one of interest (and if you insist on using an isotype control for that channel, so be it). We term these controls "FMO" or "Fluorescence Minus One" controls. (For more discussion of the need of FMO controls, see my paper on Compensation in the upcoming issue of Cytometry). Staining controls are very difficult to generate. In general, the best control for antibody binding is a cell that is exactly the same as your cell of interest, but lacking the antigen of interest. Of course, this is rarely achievable. However, one will often find very similar cells that meet the bill. In immunophenotyping of peripheral blood, you can use "nonexpressing" cell types as internal controls (i.e., naive T cells can serve as a control for measuring activation markers on memory T cells). Of course, you need to be careful, because some "nonexpressing" cells actually express the marker. Isotype controls have their place. However, most people don't use them properly. In general, I counsel people NOT to use isotype controls, but rather to use their brains to come up with a set of appropriate negative controls (which MUST be included in all experiments, as others have noted). Blind reliance on isotype controls is one of the most common mistakes in publications--and leads to the erroneous placement of gates. In any case, you are correct that investigators need to be educated more. This is one of the discussions that pops up every few years on the mailing list; perhaps it's time to have a FAQ's page (no pun intended!) on the Purdue site which includes the various discussion points, and rather than coming up with a conclusion, this page can simply serve to put forth various peoples' views so that researchers can judge for themselves whether or not isotype controls are useful. mr (PS, there is no such thing as "bad data", only "bad interpretation of data."). >It is very important to have an isotype control. I have come across some >investigators who have not being using isotype controls. I have seen some >using only unstained cells as negative controls which is also wrong. It is >like doing any other assay without a negative control. When we have negative >or non-specific binding control in other assays (ELISA, RIA etc) how can you >not have isotype controls in flow cytometry. It also relates to the recent >discussion we have been having on this forum regarding bad data. I think >investigators need to be educated more. > >Indresh Kaur Ph.D. >Phone: 281-483-8791
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