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Mario-
Several years ago (when we were switching over to staining in 96 well
plates) we did titration curves like the ones you mentioned. As
you indicate, given equal ab concentration, we did not notice a
diminution in intensity when 10E6 or more cells were stained
per well (curves were essentially superimposable over a wide range of
cells/well). Since vendor supplied protocols usually specify an
antibody volume and total reaction volume (and since most allow about
2-fold leeway) we usually find it satisfactory to scale down the
volumes proportionately (and not worry about the cell concentration
unless it is extreme). The volume of PBS remaining on cells
centrifuged in 96 well plates, "flicked" and blotted is just over
10uL/well; so 1-2 uL of stock ab is usually overkill.
This observation has saved us countless $ over the years.
We use little more antibody for a sort than we do for analysis.
The motto: keep your pellets dry and your volumes low.
Albert and Vera Donnenberg
University of Pittsburgh
Albert D. Donnenberg, PhD
Associate Professor of Medicine
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