Schwann cell (SC) transplantation is a promising strategy for axonal regeneration in the nervous system. Identifying the grafted SCs is an important aspect of this approach. The current study sought to establish a simple, reliable, fluorescent labeling method for SCs with a lipophilic molecule, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Human SCs were incubated with varying concentrations of CFSE for different time periods. Based on the viability of labeled SCs and its plating efficiency, 1 min incubation with 5 microM CFSE at 37 degrees C was selected as the optimal labeling condition. Flow cytometric analysis and fluorescence microscopy demonstrated that the fluorescence of labeled SCs would fade over 4 weeks. Immunostaining for the phenotypic expression of SC markers, including S100, GFAP, P75, and MHC-I/II at 1 and 4 weeks after incubation with CFSE showed no difference between labeled and non-labeled SCs. Mixed cultures of labeled human SCs and rat SCs for 48 h were performed in triplicate and demonstrated that no leakage of dye from labeled SCs in cell culture occurred across species. A total of 14 injections of 2x10(5) labeled SCs were performed within the spinal cord at T8 and/or L1 level in 9 nude rats. The animals were euthanized at 1 (6 injections) and 4 weeks (8 injections). Grafted labeled SCs survived for at least 4 weeks, and could be easily recognized in the nude rat spinal cord without leakage of dye to surrounding cells. The SCs migrated in white and gray matter 3-6 mm away from the injection and in the central canal for up to 12 mm. These results suggest that CFSE can be used as a fluorescent tracer of human SCs for both in vitro and in vivo studies, for a period of at least 4 weeks.