CFSE

Carboxyfluorescein succinimidyl ester (CFSE) is a fluorescent dye widely used to label live cells for tracking proliferation and lineage in flow cytometry and fluorescence microscopy. It is commonly supplied as the diacetate form (CFDA-SE), which readily crosses cell membranes.

Once inside the cell, intracellular esterases cleave the acetate groups to generate CFSE, a reactive succinimidyl ester that forms covalent bonds with intracellular amine groups on proteins. This creates a stable fluorescent signal that is retained within the labeled cells for extended periods. Because the dye is divided roughly equally between daughter cells during cell division, the fluorescence intensity halves with each round of proliferation, allowing researchers to quantify cell division by flow cytometry or microscopy.

CFSE has bright green fluorescence with excitation around 492 nm and emission near 517 nm, making it compatible with standard flow cytometers equipped with blue (around 488 nm) lasers. It is often used for tracking lymphocyte proliferation, stem and progenitor cell fate, and other proliferative processes in vitro and, with appropriate controls, in vivo experiments. Variants and related dyes in the same family (such as CellTrace dyes) provide additional spectral options for multicolor labeling.

Limitations to consider include potential cytotoxicity at high labeling concentrations, dye leakage or transfer to phagocytic cells, and signal dilution over multiple divisions reducing detectability after several rounds. CFSE labeling requires careful optimization of concentration and incubation time to balance robust signal with cellular health, and appropriate compensation is needed when used alongside other fluorescent markers.