Hi everyone,
I am struggling right now with the BrdU assay using MCF-7 cells.
We are using the BrdU assay already for a while in our lab but for measuring proliferation in PBMCs (suspension cells). This works completely fine.
Now we would like to determine cell proliferation in MCF-7 cell (from human carcinoma = adherent cells). According to the protocol (Roche), also this should work - however, after 4 h of BrdU incubation we detect yellow precipitations but only in wells in which we expect higher proliferation. It is not possible to solve them with more acid or scraping. It seems as a satuaration is reached, but absorbance values are not that high (max. of 0.8). So, the absorbance values do not increase although we can visually see a higher proliferation rate.
Has someone similar experiences or any idea what we could try?
Thanks in advance!
Hi Barbara,
we have used BrdU assay for MDA-MB-231 cells (adherent breast cancer cell line) without any problem.
The reason for lower absorbance could be precipitation. But why did the precipitation occur? Perhaps you had too much of the product. You can reduce the amount of the product by reducing the number of cells, by reducing incubation time with BrdU or simply by stopping the reaction sooner.
You can also check expiration date of the kit and proper storage of reagents (especially substrate) to eliminate the possibility that there was something wrong with the reagents.
Good luck and let us know if you find a solution :)
Klemen
Hi Klemen,
thanks for sharing your experiences!
May I ask you how long do you incubate the cells in total (we do 8 days)? How long do you incubate with BrdU (we do 4 hours)? How many cells do you seed out in a 96well plate (we do only 5000 cells/well in 96well)?
Expiration and storage of the kit should be OK.
Next time, we will try to increase acid concentration and maybe shorter incubation time... but if you like, I can update you on our findings :)
By the way: we made some picture - I will load them up. There you can clearly see the precipitation in the wells.
Barbara
Hi Barbara,
we, too, were seeding 5000 cells/well in 96-well plates, but we cultured the cells only 2 days before performing BrdU assay. During this time, the number of cells increased approximately 3-fold. Our incubation with BrdU was 1 h longer (5 h).
Increasing volume of acid might help a bit, however, I think that reducing incubation time with BrdU and/or reaction time will have more effect. Reducing incubation time with BrdU has additional advantage. If your cells proliferate rapidly and use up BrdU in the medium you loose linear relationship between the proliferation and signal. Reducing incubation time with BrdU reduces the chance that cells use up BrdU and therefore can give more reliable results.
By the way: Are your cells still growing in a monolayer after 8 days or are they growing in more than one layer? I am actually surprised that you still see proliferation after such a long time :)
Klemen
HI Klemen,
attached you find a picture of our plates - not sure if you can see the precipitation so good.
Thanks again for your suggestions - reducing BrdU is a good idea. And just FYI: we found another discussion on reserachgate about TMB substrate and precipitation (https://www.researchgate.net/post/My_TMB_substrate_for_ELISA_is_forming_black_precipitate_about_10_mins_after_stopping_how_can_I_prevent_this). So maybe that might be one parameter we can adapt. Starting with changing only one parameter, we decided now to reduce TMB subtrate, and to measure the blue color change and then, after stopping the reaction, the yellow color. Lets see...
Regading your last question: yes, they do grow in a monolayer, because we let them starve, so they grow really slow. In other literature it is quite common to incubate those type of cells for such a long time.
Best regards, Barbara
Thanks for the pictures. Unfortunately I can't see the precipitate, but I believe you that you saw it :)
Thanks for the explanation about the cells and for the link. I think any cell number is fine as long as the cells are not super dense or grow in more than one layer at the end of the experiment.
I think measuring first blue color and then yellow color is a great idea, however, I wouldn't reduce the substrate. If you don't have enough substrate, the reaction will slow down or even stop in wells with the highest cell proliferation while it will still go on in wells with lower cell proliferation, which will give you wrong results.
One additional suggestion. After adding the substrate, you can put the plate in the microplate reader and measure absorbance of blue light at several time points and then choose optimal time point for data analysis.
Just don't forget to change the color filter when you go from measuring one color to measuring another color :)
Good luck!
Klemen
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