CFSE is cell-membrane permeable and readily accumulates inside viable cells where it covalently attaches to intracellular proteins (Fig. 1). Hydrolyzed CFSE emits fluorescence and covalently attached fluorescein molecules do not leak from cells. CFSE-labeled cells can be monitored over several weeks in vivo. Therefore, CFSE is utilized for detection of viable cell as well as for the long-term observation of cell activities by fluorescent microscopy. The excitation and emission wavelengths of CFSE-labeled cells are 500 nm and 520 nm, respectively. CFSE-stained cells are shown in Fig. 2.

Fig. 1 Cell staining mechanism

Staining Procedure1. Prepare 1 mM CFSE solution with DMSO. Dilute it to prepare 10-50 μM CFSE solution with PBS or an appropriate buffer.2. Add CFSE solution with 1/10 of the volume of cell culture medium to the cell culture.3. Incubate the cell at 37ºC for 15 to 30 min.4. Wash cells twice with PBS or an appropriate buffer.5. Observe the cells under a fluorescence microscope with 490 nm excitation and 530 nm emission filters.

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Fig. 2 Cell staining with CFSECell type: HeLa