We had a similar discussion some time ago. Excess of antibody is one thing, affinity and mass transport limitation another. Just think about having 10^6 cells in a pellet of approximately 60ul and you add 1ug of antibody. You can then calculate the excess of antibody (probably better than I did last year with swapping exponents) depending on the density of binding site. Now imagine the extreme on the other end. Use a bucket and add 10^6 cells. Than you add 1ug of antibody and try to predict the resulting fluorescence or required staining time. As a simple, so not ideal solution, you can add a home made non fluorescent anti-mouse bead to capture your antibody. If it's fluorescence from captured anti mouse antibody reduces you have run out of it. Regards Gerhard -----Original Message----- From: Plett, P Artur [SMTP:pplett@iupui.edu] Sent: Friday, March 16, 2001 5:14 PM To: Cytometry Mailing List Subject: RE: Antibody concentration Hello flowers, especially those interested in the temperature-antibody incubation discussion. First of all, thank you for those very educational essays on this issue. Now, since the "golden standard" of 1 ug/million cells, which of course we all know is a titreable industry standard, is expressed in amount of antibody/cell: Question 1: how important is the total amount of antibody? Can cells influence the concentration of antibody in the solution by having it bound to the cells (if the antibody isn't in a 100-1000 fold excess)? As mentioned with the parenthetical comment "within reason" this is a frequent situation with sorting tubes where one is staining up to 10^8 cells and one really doesn't want to use up half the vial in one shot. On a more personal matter, in some experiments I did, I expressed the amount of chemokine receptor staining as 1ug/10^6 cells, which was done in the same volume (round bottom-tube dry pellet), but with different amounts of antibody depending on the number of cells that were required for the experiment. Question 2: is it appropriate to express it that way (ug/x-cells)? Question 3: is it valid to compare results if in one experiment 100,000 cells were treated this way (0.1ug/50ul), but in the next experiment 5 million cells (5ug/50ul) were incubated identically? We think that the amount of antibody (or is it concentration)plays a role. From some in vitro data we have it seems that the effect we're looking at was there in both groups, but in vivo data has been very variable (which of course is characteristic for in vivo data anyway).... thank you for any input and excuse the "multiparameter" question :) @@@@@@@@//////???????? Artur Plett, PhD Post-doctoral fellow IU School of Medicine Div. of Hematology/Oncology Indianapolis, IN 46202 -----Original Message----- From: Donnenberg, Albert [mailto:donnenbergad@MSX.UPMC.EDU] Sent: Thursday, March 15, 2001 12:22 PM To: cyto-inbox Mailing List <cytometry@flowcyt.cyto.purdue.edu> \"Cytometry Mailing List\"\"" Subject: Antibody concentration 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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