Dear Fellow Researchers,
I'm having some troubles with BrdU assay. I provide you my simplified protocol and images in attachments below. I am not able to count proliferating cells beacuse of these blurry green shapes that do not locate in the nucleus. It worked several times with PC3 cell line but from some time I'm having troubles with other cell lines (BXPC3, HCT, HT-29). Could you please tell me what should I check?
Protocol:
1. Add BrdU stock (4ul/l) to the medium for a given time (usually 24h). Final concentraton 10 uM.
Next day
1. Remove the cell culture medium.
2. Wash the cells with warm PBS.
3. Fix the cells in 70% cold ethanol (keep in room temperature for 20 min).
3. Wash the cells witt PBS (5min, RT)
4. Wash the cells with PBS + 0.5% triton X100 (2x5min, RT)
5. Incubate with 0,5 ml of PBS and 0,5 ml of 4N HCl - incubate 30 min in RT
6. Wash the cells twice with PBS (2 x 5min)
7.Incubate in 1 ml sodium borate 0.1M (1min)
8. Wash the cells twice with PBS.
9. Incubate with 1st ani-BrdU (mouse) 10ul/ml in PBS+1%BSA+ 0.5% tween (1h, RT, dark)
10. Wash the cells with PBS+0,5% tween twice (2 x 5 min)
11. Incubate with 2nd anti-mouse 1:650 in PBS+1%BSA+0,5%tween (1h)
12. Wash cell with 2 x PBS+0,5% tween (2x 5min)
13. Incubate with DAPI (15 min)
14. Wash the cells with 1 x PBS (RT, 5min)
15. Mount coverslips with fluoromount G.
Dear Mateusz Kciuk many thanks for your interesting technical question. As an inorganic chemist I'm far from being a proven expert in this field of research. However, I can suggest to you some potentially helpful literature. For example, please have a look at the following relevant link:
Top Tips for Troubleshooting Your BrdU Blues
https://www.bio-rad-antibodies.com/blog/troubleshooting-brdu-blues.html
It might also be helpful to have a look at the answers given to this closely related RG question:
Has anyone experienced problems with BrdU staining the cytoplasm instead of the nucleus?
https://www.researchgate.net/post/Has-anyone-experienced-problems-with-BrdU-staining-the-cytoplasm-instead-of-the-nucleus
(9 answers)
I hope this helps. Good luck wth your work! 👍
You seem to have background fom antibodies. You need optimize the antibody staining procedures for each cell line.
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