Tuesday, 2 November 2010

GGS LIVE - How to measure DNA synthesis in vivo?

Hello Biofreakers,

Today in the GGS LIVE section we are going to look at techniques that allow to observe cells that replicate DNA (are in the S-phase of the cell cycle).

Method: Labelling and detection of replicating (aka. S-phase) cells.

About: Both methods that will be discussed (flow cythometry and immunofluorescence) use specific derivative of nucleotide that is incorporated into DNA during the S-phase of cell cycle. Later this modified nuclotide can be detected and S-phase cells analysed.

What: Labelling of chicken DT40 S-phase cells.

One of the most common nucleotide derivative used in cell biology is a bromodeoxyuridine (BrdU, for structure and other applications go here Meet the chickens - sister chromatid labelling). BrdU pulse will result in its incorporation into cells DNA. In our case study such cells will be analysed by flow cytometry and immunofluorescence. This techniques are very different but sample preparation is very similar, see picture below.

 And this one:)

Similarly like in the Western and Southern Blotting, antibody used here is conjugated to a moiety that gives a specific signal, in this case antibody is conjugated to a green fluorophore called FITC. The signal from fluorophore can be detected in many different ways. In our study case, cells designated for flow cytometry will be analysed with flow cytometer:) and ones on the slide will be analysed with fluorescent microscope. Ok lets check the results of our experiment.

Lets look first at the panel A where S-phase labelled cells are analysed by flow cytometry (for those that are not familiar with flow cytometry, please visit this post GGS LIVE - flow cytometry). The first histogram in the panel A represents a DNA content profile. On the x-axis we have a DNA content. The first big peak are cells in G1 phase of the cell cyle with 2N DNA content, second smaller peak are cells in G2/M phase of cell cycle with 4N DNA content and finally all cells between those two peaks are cells in S-phase with DNA content between 2N-4N. On the y-axis we have counts (the BrdU incporporation is only relevant for the other two dot plots). So you can easily see that the most number of the cells is in the G1 phase of the cell cycle followed by S-phase and G2/M cells. This histogram is often called one dimensional cell cycle plot as it measure only DNA content. The other two dot plots are often called two dimensional as we measure DNA content and BrdU incorporation. What you can see on the first dot plot is exatly the same as on the first histogram but represented in a slightly different way. Now, there is no peaks but each dot represents a single cell. Similarly the first bunch of dots (cells:) is population of G1 phase cells and the other bunch is population of G2/M cells. Everyhting between them are S-phase cells. Look know on the last dot plot. The situation is similar but these cells were pulsed with BrdU and what you can see know is that S-phase cells did shiftet to upper values on the y-axis (which is a measure of the fluorescence that is directly proportional to the antibody that recognise BrdU). This picture is often called a horseshoe.

On the panel B we can see pictures of cells. On the first panel we see DNA that was labelled with DAPI (blue flurophore), in the middle we see a replication foci that were lebelled with BrdU and detected with antibodies. On the last picture we see an overaly of the two, which clearly indicates that replication foci are localised to DNA (what makes sense:).

Both techniques are very useful in analysis of replicating cells. The flow cytometry allows for a very robust and quick analysis of S-phase cells (be aware that flow cytometry allows for quantification of the cells in each phase of cell cycle). We can use this technique to monitor S-phase cells for example after treatment with different drugs. Immunofluorecence method does not have power of numbers but allows detecting of for example other proteins colocalization to replication foci (if protein X will be labelled red we can see if it colocalizes with replication foci). Both techniques are very powerful and are commonly used in each cell biology laboratory.

I hope u enjoyed:)



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