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 Transcription Factor 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. Each vial of CFSE may be reconstituted to a stock concentration of 10 mM with 90 µL of anhydrous DMSO; once reconstituted it should be used within 6 months and protected from light and stored at -20°C with dessicant; avoid freeze-thawing.