Chapter 17 Flow cytometric analysis of cell division history using dilution of Carboxyfluorescein Diacetate Succinimidyl Ester, a stably integrated fluorescent probe

This chapter reviews the use of a technique based on the serial dilution of a stably binding intracellular fluorochrome, carboxyfluorescein diacetate succinimidyl ester (CFSE), which allows eight to ten sequential cell divisions to be analyzed by flow cytometry. When incubated with cells, the fluorescein-based CFSE crosses the cell membrane and attaches to free amine groups of cytoplasmic cell proteins. After enzymatic removal of carboxyl groups by endogenous intracellular esterases, CFSE acquires identical spectral characteristics to fluorescein, with optimal excitation by 488 nm argon laser light, emitting strongly at 519 nm, and as such is compatible with almost all single and multiple laser flow cytometers. On cell division, CFSE is distributed equally between progeny, allowing the division history of a cell population to be determined. This technique can be used to investigate the behavior of cells in vitro, as well as division of transferred cells in vivo. A major advantage of the CFSE based technique is that viable cells from defined division cycles can be recovered, allowing functional characteristics to be related to differentiation stage.