Hello Michal, Negative gating can be very useful in multicolor applications, in that it capitalizes on the propensity of nonspecific staining to occur with all the antibodies in your staining cocktail (this assumes that your antibodies are all near their respective optimal concentration, and that none of them are aggregated). The general notion that seems descriptive is that most cell preps have some cell debris, and that this debris can stick to cells and to antibodies. To the extent that this problem contributes to your "background," you can magically gate it away by focusing your attention on the cells that didn't bind one of the antibodies in your cocktail, a "negative gate." Sometimes referred to as a "dump channel," lots of investigators simply add an irrelevant antibody with a fluorchrome that is not being used in the important part of the assay. Like the specific antibodies, this irrelevant antibody will bind to the debris, and applying a negative gate on this irrelevant antibody will filter out cells which have this kind of background staining, and sometimes pretty miraculously turn a mess into a publication. The most common implementation of a related strategy is to gate out dead cells which stain with propidium iodide. Dead and damaged cells are notorious for nonspecific staining, and a PI-negative gate is very generally used. Even in applications where the PI stain will contaminate other important red signals, the PI+ cells are much brighter than most immunofluorescent signals, and you can use a PI-bright gate to generate meaningful data about the dimmer, healthy antibody-PE positive cells. The utility of these tricks with tetramer staining makes sense and ought to work, but it might be worth thinking about. You're using labeling reagents with pretty different protein structure and molecular weight, maybe different kinds of irrelevant binding features, storage buffers, etc. Note that any gating strategy can be a complicated filter, and unless you're pretty sure you understand and control for subtle effects, it's certainly caveat emptor. I don't think it's really very different from filtering a clumpy sample with 70um mesh...you never really know what kind of bias you have introduced to the sample, so that interpretations of the frequency of a given population can be complicated. The best advice is certainly to use sample prep strategies that minimize debris in the first place. JD>
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