CFSE is widely used for cell tracking and proliferation studies. It has also been used in CTL assays and cell motility studies. CFSE readily crosses intact cell membranes. Once inside the cells, intracellular esterases cleave the acetate groups to yield the fluorescent carboxyfluorescein molecule. The succinimidyl ester group reacts with primary amines, crosslinking the dye to intracellular proteins. Cell division can be measured as successive halving of the fluorescence intensity of CFSE. Cells labeled with CFSE may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers, such as the Foxp3 Staining Buffer Set (cat. 00-5523) or the IC Fixation Buffer (cat. 00-8222) and Permeabilization Buffer (10X) (cat. 00-8333). CFSE has a molecular weight of 557.47. After the acetate groups are cleaved, it has a peak excitation of 494 nm and peak emission of 521 nm. CFSE should be reconstituted in DMSO once reconstituted it should be protected from light and stored at -20°C avoid freeze-thawing.