BrDU proliferation assay

[Dennis Goode]

chris at austin.unimelb.edu.au
on 23 Feb 95 17:34:58 wrote:

> Has anyone had experience with the BrDU labelling assay
> for cell proliferation?  I have attempted to use it in 
> a manner analagous to the continuous labelling of cells
> with tritiated thymidine.  This a simple method giving
> much information of cell cycle time etc.  However, 
> rather than getting a simple increase in labelling over
> time with an eventual plateau when all cells are 
> labelled, I obtained a sine curve with two peaks, the 
> second less than the first.  I would be glad of any 
> explanations (or even half convincing fictions!).
> 
> Thanks in advance,
> Chris Bradley
> 
Chris:

We have used the BrdU labelling assay procedure in the continuous 
labeling mode in a manner similar to the 3H-TdR continuous labelling 
method (Goode et al. J. Cell Biol. 79: 67a). We get first order curves 
that plateau at approx the percent of proliferating cells.
If you get steps in your data, that indicates some synchrony of your 
cells or a daily or otherwise cyclic peak of entry into S of your 
cells. However, theoretically your data should never go down if BrdU 
is present continuously. What you may have done is to add BrdU in low 
amounts that are incorporated into your cells essentially as a pulse, 
leaving no label for further incorporation. Thus the % labeled cells 
can go down as unlabeled cells pass thru M and divide, thus diluting
the fraction of labeled cells. The % of labeled cells will then 
increase again some time later when the labeled cohort of cells have 
gone thru a cycle and divided again, essentially giving you results 
with two peaks that look like the Fraction-labeled Mitoses method 
after pulse labeling.  

In culture with synchronized cells, we see a stairstep pattern, but 
usually a smooth function with unsych. cells. We even have students 
do this in our undergraduate cell biology labs and they get good 
results, so something funny is happening in your system. Is it in 
vivo or in culture? In vivo injections of TdR or BrdU are essentially 
pulses; to label continuously in vivo, you need something like an 
osmotic pump.

I hope these suggestions help.

Dennis Goode            "Study cells, not gels"
Dept of Zoology          Graffiti on mens room wall
Univ. of Maryland        MBL, Woods Hole MA
College Park, MD 20742
(301)405-6935