Objective: We have shown that buccal epithelial cells (BEC) from humans can contain a polymicrobial intracellular flora. Members of that flora can induce proinflammatory responses. However, our subjects all had healthy oral mucosa. This might reflect tolerance of bacterial invasion by live BEC. Alternatively, inflammation might not occur if invaded cells were mostly dead, and thus unable to mount a response. This study addressed that issue, by determining the vital status of BEC and the bacteria associated with them. Design: Initial experiments indicated that BEC were anomalously permeable to the DNA stain propidium iodide. We used that property to develop a protocol that combined the DNA stains SYTO® 9 and propidium iodide (indicators of bacterial viability) with the esterase substrate calcein blue AM (an indicator of BEC viability), and Annexin V Alexa Fluor® 647 conjugate (an apoptosis marker). That protocol was applied to BEC collected from 36 human subjects. Results: On average, 70% of BEC displayed calcein blue staining, with no binding of Annexin V, 25% showed signs of apoptosis, and 5% did not stain with calcein blue. The mean percent of BEC with live cell-associated bacteria was 29%. Collectively, 25% of total BEC displayed calcein blue staining and live (SYTO® 9 stained) bacteria. Only 1% of total BEC were negative for calcein blue and associated with live bacteria. Conclusions: Our findings suggest that live BEC are tolerant of bacterial invasion. This may be due to complex interactions between members of the polymicrobial flora and their host BEC.
- Buccal epithelial cells
- Vital stains