[Flow cytometry cell-cycle analysis for in vivo corneal epithelium]

To investigate the cell-cycle kinetics of in vivo corneal epithelium, S phase cell analysis using either bromodeoxyuridine or 3H-thymidine incorporation and mitotic counts on flat preparations have been used extensively. This type of analysis represents S-phase cells, but not G0, G1, or M-phase cells. In this study, in vivo and in vitro rabbit corneal epithelial cells, in which the nuclei were stained with propidium iodide, were analyzed by flow cytometry to observe the overall distribution of each cell cycle. The method requires single cell suspensions from corneal epithelium. For this, an intact sheet of corneal epithelium obtained using low concentration of neutral protease, dispase II, was treated with trypsin and EDTA. The present results indicated that in in vivo corneal epithelium 97% of total cells were in the G0/G1 phase, 2% in the S phase and 1% in the G2/M phases; while in the in vitro epithelium 71% of cells were in the G0/G1 phase, 14% in the S phase and 15% in the G2/M phase. Although the present method can be applied to observe the cell-cycle in in vivo epithelium if used properly, methodologies to maintain single cell suspensions are required for future studies.