Autofluorescent characteristics of human periodontal ligament cells in vitro

The periodontal ligament (PDL) is a connective tissue surrounding the tooth root and attaching it to its bony socket. Being the predominant cell type of this connective tissue, PDL fibroblasts are thought to be responsible for PDL homeostasis and regeneration. Autofluorescence is found in abundant natural substances and many cells exhibit some intrinsic level of autofluorescence depending on the cell type and function. Thus, autofluorescence often makes immunofluorescence staining and flow cytometric analysis difficult. Therefore, in the present study, we have investigated the autofluorescent characteristics of human periodontal ligament (PDL) cells and compared them to cells of epithelial and mesenchymal origin. The results of fluorescence immunostaining and flow cytometric analysis revealed that PDL cells exhibit an extremely high level of autofluorescence in the short wave length region including FITC emission spectra which exceeded those of HEK293 cells, oral keratinocytes and MG63 osteoblast-like cells. This observation reflects the high capacity of PDL cells to synthesize extracellular matrix proteins and collagens. In the long wavelength region, the autofluorescent intensity of PDL cells was the lowest amongst the investigated cell lines. A permeabilization step with Triton X-100 also reduced the autofluorescence of PDL cells in the FITC channel. Our data indicate that conjugated antibodies with emission spectra in the red fluorescence region might be the proper choice for studies using immunofluorescence staining and flow cytometric analysis of human PDL cells. Fixation and permeabilization of the cells might also be beneficial in this respect.