From: Albert D Donnenberg, PhD (donnal@novell1.dept-med.pitt.edu)
Date: Tue Jun 18 1996 - 18:23:09 EST
> Date: Mon, 17 Jun 1996 08:45:05 -0700 > From: Mario Roederer <Roederer@Beadle.Stanford.EDU> > Subject: Antibody concentrations > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> 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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