Calman, I've spoken out against using isotype "matched" controls many times, and won't go into that again, except to say, don't use them for antigen density measurements! Instead, use a parallel aliquot of cells that are stained with everything EXCEPT for the color on which you are doing the measurement (and leave that one unstained). It is very important to include the other antibodies, not just isotypes for them--to fully correct for problems with compensation, spectral shifts, etc. Eventually, you should subtract the background value. But this assumes that you are only interested in a single value, and single values are not good for representing distributions (unless the distribution happens to be normal, or, more precisely, log-normal). Our approach was to calculate a series of values, including not only the median (which is far better than the mean when you are using log-scale), but also the 10th, 25th, 75th, and 90th percentiles of fluorescence. This allowed us to build up a picture of the distribution of fluorescences. Correcting these values by background, however, is not simple. i.e., from the 10th percentile, do you subtract the median of the unstained? the 10th percentile of the unstained? Once you start considering questions like this, you realize that subtracting the MESF of the control from the MESF of the sample has significant pitfalls of its own! Our approach now is to use the calibration platform in FlowJo--this platform allows you to rescale any fluorescence channel based on a standard fluorescence measurement (either by fitting to a bead set or just by entering the appropriate conversion value). At this point, all graphs & statistics are shown in terms of absolute molecules rather than relative fluorescence. Thereby you can easily calculate the above statistics (or show histograms etc) in units which are meaningful for your final desired answer, and you can then decide what type of analysis is appropriate. So, while the simplistic answer to your question is "Yes, subtract background values", there is an enormous complexity underlying the process which really requires considerable more thought. mr At 11:42 AM -0500 1/6/00, Calman Prussin wrote: >We are quantitating cell associated antigens using standardized beads and >then generating an MESF from the standard curve generated from the beads. >The question has come up whether or not to "correct" the MESF values >obtained from the specific antibody by subtracting the MESF values obtained >for the isotype matched controls. > >The problem with correcting the values is that it has little effect on the >samples with high density of Ag, but has a large effect on those with low >expression. As such, if we run replicate samples on the low expressing >samples we find a larger amount of variation in the corrected MESFs. My >bias is not to use it, as it seems to introduce more "noise" into the >system. Your thoughts? > >Thanks! > > > _______________________ > > Calman Prussin > > Laboratory of Allergic Diseases > > NIAID/ National Institutes of Health > >
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