Cell Cycle Analysis - Bromodeoxyuridine
The limitation of looking at a single fluorochrome is that we don't get any kinetic information - we don't know that the cells that have an 'S Phase' amount of DNA are actually cycling. To assess this we can use bromodeoxyuridine (BrdU).
BrdU is an analogue of thymidine and will be taken up into the DNA of cycling cells. To detect this, we can unwind the DNA (by using acid, alkali or enzyme) and then use an antibody against BrdU. In this way, we can separate G1, S and G2 cells.
Obviously BrdU-positive cells will equate to S phase cells only when the labelling time is short as cells in late S at the beginning of the labelling period may pass through to G2 by the end of labelling. However this does mean that we can answer different questions by varying the labelling time with BrdU.
To compare the rate of cycling between cell types or different treatments of the same cells, we generally use a 30-60min labeling period.If we want to assess transition times through the cells cycle compartments, we can use a pulse label - a very short (2-3min) label, then wash the cells and sample at various time points. In this situation, only cells in S will be labelled and we can follow their progression through the cycle as in the example below.
Click on the image to view diagram in full size
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There are two major protocols used in this Lab based on unwinding the DNA by acid or by enzyme (DNase) and these are available below:
More recently we have used EdU (5-ethynyl-2'-deoxyuridine) incorporation which uses a copper catalysed 'click reaction' to label cells by the addition of a fluorescent azide compound onto the incorporated EdU. A protocol in pdf format is available here.
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