could any one please tell me a good protocol for BrdU of MG,, especially these points.
(1) how to plate cells, number, time of plate & FBS conc.
(2) what is the suitable time for incorporating BrdU,, is it overnight or only few hours,
(3) should I put BrdU simultaneously with the drug or after finishing all time of treatment(24 hr)?..
thanks
Please see this link:
https://www.exalpha.com/protocols/brdu-labeling
(1) This depends on your experiment, there is no one answer.
(2) Different times will tell you different things. If you are interested in acute proliferation (mitotic index etc), 6 or so hours immediately before fixing will do just fine. If you are interested in survival etc, a pulse days before may be required. In general, the shortest time you can get away with is the best, as BrdU is not great for cells. Also keep in mind that the halflife of BrdU at 37C is only a few hours anyway.
(3) As per 1 and 2, this depends on what you are looking at.
As a general note, if you can afford it, EdU is a much easer alternative, as it's much easier to detect.
I agree with Niels - EdU is much better for both the cells (its smaller than BrdU) and for ease of downstream analysis (no work to expose BrdU and so you can co-stain with other antibodies). Life technologies do a good kit for this.
As for timing, it completely depends on what your question is but again bear in mind the half life, so if you want to do a longer term chase you may have to apply more.
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