reproducibility with anti-BrdU Abs

Kevin S Sweder kevin.sweder at bms.com
Fri Nov 18 11:00:54 EST 2005


Dear colleagues,

We are determining CHO cell cycle kinetics using BrdU, FITC-conjugated 
anti-BrdU antibodies, and propidium iodide. We have had good luck with 
our bivariate analyses, but recently we suddenly have been unable to get 
clear and consistent results. Eliminating variables resulted in 
identification of a particular lot of antibody from a well-known vendor 
was not working as advertised. Instead of getting a nice "horseshoe" 
distribution for growing cells, we obtained a shift in all cells in the 
population to a brighter fluorescence in the FITC anti-BrdU channel 
almost to the level of S phase cells in past experiments (see below). 
Side by side comparison of this antibody to antibody from another vendor 
clearly indicated that the antibody from the first vendor was not 
functioning. Has anyone else had a similar experience and are there some 
vendors who have a problem with quality control?

Sincerely,
Kevin Sweder

For the above figures:
Upper left - 1:25 dilution of PRB-1 (anti-BrdU 1° Ab and Alexafluor 
488-conjugated 2° Ab)
Upper right - 1:50 dilution of PRB-1 (anti-BrdU 1° Ab and Alexafluor 
488-conjugated 2° Ab)
Lower left - 1:50 dilution of B44 (FITC-conjugated anti-BrdU 1° Ab)
Lower right - 1:50 dilution of CD4 (FITC-conjugated anti-BrdU 1° Ab)

All samples were from the same vehicle-treated CHO culture.
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