FLOWers- I would like to emphasize a practical point arising from Simon Hunt's excellent disquisition on antibody binding (buried somewhere in the middle). It's the Ab concentration that matters and not the amount of Ab per cell! The practical ramification is that if you stain in microtiter plates, the volume of a dry cell pellet (as dry as you can get it by flicking and blotting) containing 100,000 to several million cells is only about 12 microliters. Thus you can use about a tenth of the amount of Ab recommended by manufacturer's protocols that advise resuspension in 100 microliters. Practically we use from 1 to 3 microliters of antibody per test, depending on how a specific antibody titrates and on how much the antibodies dilute each other in multi-color combinations. It also follows that (within reason) a very large number of cells can be stained in a very small volume with little reagent. This is helpful for sorting. Albert Donnenberg
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