Cell Proliferation Round 2 And Beyond: The Dye Dilution Method

Like the legendary fight between boxers Bowen and Burke in 1893, the cell cycle in some cells goes on and on..round 1, round 2, round 3, round 4…before the final bell is rung. Nucleotide analogs, like BrdU or EdU, are great for examining 1-2 cell cycle division(s).  In many studies though, for example the proliferation of T cells following a specific stimulus, it is desirable to look at cells over more divisions. Typically you may want to follow cell proliferation over 96 hours or more in which time they will have undergone 5 or 6 divisions.  In these cases a dye dilution method is used.

The dye dilution method

The principle of the approach is simple – cells are labelled with a fluorescent dye that binds to either lipid membranes or cellular proteins. When a cell divides, it shares the dye equally between daughter cells so each of these will have half the amount of total fluorescence of the parent cells. When the daughter cells divide, they will have a quarter the fluorescence of the original cell and so on. The number of divisions that can be measured is only limited by the dynamic range of the detection system and the fidelity of the way the dye is shared. The protocols used are very simple and well established.  Generally you stain  your cells and then stimulate them in some way – the success, or not, of the stimulus is determined by the number of division peaks after a defined time period. A typical result is shown in Figure 1.

  As well as seeing how many times the cell has divided, you can also calculate how many cells have divided, the average number of cell divisions and the proliferation index.

Proliferation dyes

There has been a proliferation of proliferation dyes in the past few years – which one best suits your experiment? It will, as always, depend on the question you are asking, but there is a range of dyes available to answer it! If all the information you seek is about cell division, then any of the dyes below can be used.   But if you also desire other information, such as expression of a marker, then there will have to be a decision as to which other colors can be used (as with all flow cytometric experiments!).

Lipophilic dyes

Lipophilic dyes (PKH dyes; CellVue Claret) bind to lipids in the cell membrane and are often used to label cells for cell tracking experiments.  Although they can be used to monitor cell division as well, the protein binding dyes are often preferred for this. The PKH dyes come in two different flavors – PKH2 and PKH67 emit green fluorescence whereas PKH26 is an orange fluorochrome.

Protein dyes

Generally the protein binding dyes (CFSE and CellTrace dyes) are preferred for dye dilution studies as the variability of binding is smaller (lower CV).  This makes distinction of the division peaks easier. CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescein derivative and fluoresces green. This is slightly limiting especially if your cells happen to express a green fluorescent protein. More recently, new protein-binding dyes such as the CellTrace range from Life Technologies and the eFluor dyes from eBioscience have been developed. These fluorochromes have a range of emission colors and may be combined with other analyses such as DNA content or surface marker expression to yield very precise information about specific subsets within a population. The dye dilution method is pretty easy, however, as with all laboratory procedures, there can be pitfalls. So stay tuned for some practical tips and tricks in the third article in this series.