The introduction of fluorescent cell staining and division tracking dyes has made it possible to monitor the number of cell divisions during proliferation, and to examine the relationship between proliferation and differentiation.

 

Fluorescent tracking dyes can be categorized into two major groups:

 

•General protein labeling

 

General protein labeling dyes such as carboxyfluorescein diacetate succinimidyl ester (CFSE) act by forming random covalent bonds with amino groups on cellular proteins resulting in a highly fluorescent membrane impermeable marker. CFSE is highly fluorescent and when excited with a 488 nm laser it emits in the "green" channel at 515 nm.

 

•General membrane labeling dyes

 

General membrane labeling dyes such as PKH and Cellvue are lipophilic compounds that stably but non-covalently insert into the plasma membrane. General membrane labeling dyes vary in fluorescent intensity and can be obtained in many colors, making them useful for multi-color experiments.

 

 

With each cell division, the dye is divided roughly equally between the two daughter cells. Therefore, the dye fluorescence intensity is a measure of the number of divisions any given cell has undergone.

 

After staining, the cell population is analyzed at regular time intervals by flow cytometry. A histogram of the number of cells as a function of dye fluorescence intensity is generated. Typically, cell proliferation dyes can be used to track up to 8 rounds of division before fluorescence intensity is reduced to the autofluorescence of unstained cells.

 

The FCS Express Proliferation Add-on provides curve fitting algorithms to estimate the proliferation fraction.

 

For more information on proliferation analysis, please see http://www.denovosoftware.com/site/ProliferationAnalysis.shtml.