Most Protocols for BrdU assay to determine cell cycle phases require PBS-Tween for washing and AB-incubation. I did the assay now for the first time and my cells (BxPC-3) got clumpy, or even lysed.
Is Tween-20 actually needed (I used 0.2%) and why?
The BrdU antibody I use is from Millipore, clone BU-1 (Cat. FCMAB101A4) and it says that it doesn't require any DNA denaturing steps, so I didn't treat my cells with HCl.
Fixation was done in EtOH.
Tween20 is a detergent to minimise non-specific binding and maybe open up the cell nucleus, but you still need to denature your DNA (with HCl), usually. Tween20 (or other detergents) can open up (or lyse) cells, and sticky DNA will cause your cells to clump. We have found that EdU assays are better (less messy) than BrdU assays where applicable. Check out EdU spec sheets at Life Technologies.
I think that the amount of Tween-20 is high in your assay. We typically use 0.05%. Try reducing the concentration. Tween-20, being a detergent, permeabilizes the cells. High concentrations can lyse the cells resulting in clumping.
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