BrdU incorporation assay
- Cells are grown on 22 mm coverslips such that they are rapidly dividing (usually 50-70% confluent).
- Incubate cells in 100 mM BrdU (Roche; stock of 10 mM in PBS) for 30 minutes to 4 hours. For longer incubations, use 10 mM BrdU.
- Aspirate medium and immediately fix for 30 minutes at –20oC with ice-cold fix (which we store at –20oC). Fix is 70 ml ethanol 30 ml glycine pH 2.0.
- Wash cells in PBS and process for immunofluorescence.
- anti-BrdU (Roche) is diluted 1:50 for use. Cells may be counter stained with DAPI and some antibodies will work for double labelling. Cover slips are mounted in Mowiol as for the standard immunofluorescence protocol.
NB At short time points, the BrdU incorporation is weak and hard to see but suitable for imaging.
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