Dr. Carl Stewart of Roswell Park Cancer Institute from Buffalo, NY will be another authority in this discussion of using isotype control. I hope to hear his input in this discussion. Arazdordi Toumadje, Ph.D. National Stem Cell Resource Cell, Molecular and Developmental Biology Program American Type Culture Collection Manassas, VA 20010 phone: 703-365-2700 ext 280 Fax: 703-365-2790 -----Original Message----- From: Ray Hicks [mailto:ray.hicks@btinternet.com] Sent: Saturday, November 03, 2001 12:47 AM To: Cytometry Mailing List Subject: Re: Isotype controls (my annual 3 cents worth on this topic) Hi Mario, Just to make the full nickel, I'll add my two cents. This is one of my bug-bears too. We actually did get in touch with a manufacturer whose isotype control antibody was as bright as a positively stained population (and brighter than the obviously unstained cells in that tube) - their recommendation was to titrate the control antibody, "Hmm" as you so rightly say. I try to get people to use isotype controls to flag problems with "non-specific" binding, then adopt strategies to avoid it (eg blocking), then apply that strategy to their experimental staining. It doesn't always happen. When we used lots of second-layer reagents, there was a great need to use single controls that had all second layers, but only one primary antibody, because the second-layers would be binding at least as much in the experimental as they would in the controls. Most reagents are direct in my lab now (an advantage, presumably, of doing less than five colours generally), and I'm not convinced of the advantage of adding isotype control antibodies into the compensation control tubes, so I'll be interested to see that paper for a more realistic alternative. If Paul Robinson is after votes on whether or not to set up a FAQ section, I'll add mine in favour, Ray > From: Mario Roederer <roederer@drmr.com> > Date: Thu, 1 Nov 2001 19:58:47 -0500 > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Subject: Isotype controls (my annual 3 cents worth on this topic) > > > 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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