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Inquiry: I am going to perform the BrdU assay on cells which are adhesive but are also quite fragile. With other similar assays I have been having trouble with variability between replicates because cells get easily detached through the process. I was wondering if I could use DAPI as a means to normalise the BrdU signal to the number of cells still present in each well (whether these are dead or alive)? Do you have a suggestions of how this would be acheived?
Asked on Apr 11 2016
The fixative solution provided with BrdU Cell Proliferation ELISA Kit (ab126556) does an excellent job of keeping the cells in the wells. So it should not be necessary to do anything further to normalize the signal to the number of cells.
That being said, it is an interesting idea to do it. If the reader can detect DAPI, it might be possible to test the DAPI with the fixing solution and substrate/stop solutions provided with the kit. The largest unknown would be how the DAPI will react with the addition of the substrate and stop solution. The substrate can be read without the stop solution, if necessary, (at 650 nm) but it will lose sensitivity.
So if you have the ability to read the DAPI and the TMB signals, you can can try it. However, this may not work and we cannot guarantee it. Therefore, if you want to try, we would recommend to build controls into the assay design and also know the number of cells that have been plated. Bad CVs can be dealt with using an increased number of replicates.
回复于 Apr 11 2016