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Proliferation Experiments

Written by Tim Bushnell, PhD

Cell proliferation can readily be measured by flow cytometry.  Depending on the research question, there are several different techniques that can be used.

Cell counting experiments

This relatively straightforward experiment where the investigator uses one of several counting techniques to see if there is an increase in the populations. This can be done using a microscope and a hemacytometer, a flow cytometer with counting beads, image based tools (like the T-20, the Countess, or the Cellometer), or coulter counter type instruments (including the Coulter Counter the Scepter and the Casy counter)

Cell cycle experiments

One of the earliest techniques developed in flow cytometry.  Using a DNA binding dye, flow cytometry can readily identify the major phases of the cell cycle.  This technique can be used in comparing samples treated with different conditions to compare how quickly cells move through the cell cycle, or if the cells accumulate in a specific phase of the cell cycle.

BrdU/EdU experiments

Using this technique, cells are labeled with a thymidine analog.  This is only taken up during the S phase of the cell cycle.  It is detected using either an antibody (BrDU) or click-it chemistry (EdU).  Measuring the cells that have taken up the BrDU/EdU, especially in conjunction with a cell cycle dye, can help provide a measure of proliferation.

Dye dilution experiments

In this technique, cells are loaded with a fluorescent  dye (such as intracellular CFSE or membrane intercalating PKH dyes).  As a cell divides, the fluorescent dye is segregated approximately equally between the two daughter cells.  This results in a decrease in the fluorescent signal.  Each of these techniques has different advantages and limitations, but these assays can typically follow 5-7 generations of cell divisions and since the fluorescence can be measured in live cells, is that specific divisions of cells can be sorted for additional analysis.

As can be seen, there are several different methods for measuring cell proliferation, all of which are readily amenable to flow cytometry, especially in polychromatic panels, allowing for the detailed dissection of the cellular processes in the context of interesting phenotypic subsets.

Tim Bushnell, PhD

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