Fluorescent labelling of MC3T3 cell line by 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate, succinimidyl ester mixed

Kook, Min Suk

Abstract

BACKGROUND: 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3.
MATERIALS AND METHODS
The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% CO2 at 37 degrees C using alpha-minimal essential medium (alpha-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 microM. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% CO2 at 37 degrees C for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability.
RESULTS
For concentration between 5 and 10 microM, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 microM. In the concentration of 15 microM., the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 microM. was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%.
CONCLUSION
These results suggests an incubation period of 15 minutes at 15 microM. of CFSE provides best labelling of MC3T3 in vitro.