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5 Mistakes Scientists Make When Doing Flow Cytometry Proliferation Experiments

Written by Tim Bushnell, PhD

Measuring cell proliferation can be done in a number of ways.

There is the below tried and true method of counting cells. This straightforward assay can help determine if the cells are proliferating and by comparing counts. Here, a researcher can determine that the experimental treatment is increasing cell growth.

A second method of measuring proliferation involves using a radioactive tracer like 3H-Thymidine. In this assay, the amount of the isotope taken up by the cells correlates to the amount of DNA synthesis, and therefore growth. Of course this requires using radioactivity and all that entails.

A third method is using a something like yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (also known as MTT). Here the MTT is reduced by the mitochondrial enzyme succinate dehydrogenase. Cells are treated with an organic solvent and the purple formazan product of the MTT is measured with a spectrophotometer.

These three methods, unfortunately, only give results of bulk population growth. Many researchers are interested in knowing how different cell populations are proliferating based on different stimulation conditions.  Some even want to isolate cells after a specific number of cell divisions. The solution to that assay is the dye-dilution method. 

This technique was first introduced in 1994 by Lyons and Parish and has heavily cited. In this technique, cells are labeled with a dye (CFSE was the first used) that intercalates into the cells in such a way that as the cells divide, the dye is segregated roughly equally into the daughter cells. As the cell divide, the dye is diluted out and by counting the peaks, (or modeling the pattern) the number of original dividing cells can be calculated.

5 Proliferation Assay Mistakes To Avoid

In order to run a successful proliferation assay, you must avoid these 5 mistakes:

1. Not knowing the qualities of the dye you’re using. 

Dyes used for proliferation must be bright, readily taken up by the cells and distributed equally between daughter cells.

CFSE and its derivatives intercalate into the cell where they label intracellular proteins. One limitation of the CFSE dye is that there is a proliferation-independent loss of fluorescence in the first 24-36 hours. Thus interpretation on early proliferation has to take this into account.

Alternatively, lipophilic dyes like PKH26, can be used. These dyes are membrane bound, and are a good alternative to the CFSE. However, they can be more tricky to label cells as cell size and mixing can affect the labeling. Unlike CFSE, there is not a loss of signal after labeling, making these dyes better for short proliferation.  If the cells are going to be fixed, it is important to avoid using organic solvents when using membrane dyes.

2. Forgetting to titrate your dye. 

Too much of a cell tracking dye can negatively impact cell function and viability. Thus, titrating to the highest level that doesn’t affect the cells is critical.  Usually this titration can be one at a fixed cell concentration – thereby reducing the complexity of the testing.

3.  Not using the proper controls.

Both positive and negative controls are critical. So too is the optimization of the instrument. Once the titrated concentration is optimized, performing a voltage optimization is critical.  Using the unstimulated concentration, place the labeled cells at the highest decade.

4.  Forgetting to account for viability or doublets. 

As with every assay, identifying the live cells are important. Cells lose the proliferation dye as they undergo apoptosis, so ensure that these cells are removed by viability. Likewise, removing doublets using pulse geometry is important to ensure that the true proliferation rates are measured.

5.  Not collecting enough cells to accurately model the data.   

Too few cells collected and the data models used for proliferation cannot accurately model the process. As with most flow experiments, more cells are better than less.

Additional References
Lyons A.B. and C.R. Parish (1994) J Immunol Methods 171:131-137

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Tim Bushnell, PhD

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