Abstract:

BACKGROUND: Chlormethylbenzamido-1, 1-dioctadecyl-3, 3, 3’, 3’-tetramethylin-docarbocyamine (CM-DiI) and 4',6-diamidino-2-phenylindole (DAPI) are two kinds of tracers which are commonly used for labeling cellular membrane and nucleus respectively.
OBJECTIVE: To investigate the feasibility of CM-DiI combined with DAPI labeling human umbilical cord blood mononuclear cells and to observe the changes in morphology and activities of the cells cultured in vitro after double labeling.
METHODS: The freshly isolated mononuclear cells derived from human umbilical cord blood cells were double labeled with CM-DiI and DAPI, and the double labeled human umbilical cord blood cells were cultured in vitro, the changes of the morphology was observed under inverted phase contrast microscope, the activity of the cells at different times was detected though Trypan blue staining, at the same time, human umbilical cord blood cells double labeled by CM-DiI and DAPI were counted under the inverted fluorescence microscope at different times.
RESULTS AND CONCLUSION: The membrane and nucleus of human umbilical cord blood cells present red and blue fluorescence respectively under different wavelengths with fluorescence microscope after double labeling by CM-DiI and DAPI for 15 minutes. The CM-DiI/DAPI double labeled human umbilical cord blood cells were in vitro cultured for 1, 3, 7, 14 and 21 days, and there was no significant difference in the number of positive cells double labeled by CM-DiI and DAPI at different time points under fluorescence microscope. The survival rates were 95.6%-98.8%. There was no significant difference in cell morphology between the in vitro cultured double labeled cells and the cells without labeling, and the growth state, adherent ability and cell proliferation ability of the double labeled cells were not changed. Human umbilical cord blood mononuclear cells can be effectively labeled by CM-DiI and DAPI at the same time. Both of the tracers have slower fluorescence decay and non-toxic adverse reactions on the living cells and are suitable for labeling the stem cells.