Figure 3

Apoptosis of human cultured gingival epithelial cells exposed to the biofilm. (A) Scheme of the co-culture design as used in both single cell culture dishes and 6-well plates. Biofilms were placed upside-down on a ring support that was layed onto the gingival epithelial cell layer. (B) TUNEL labeling of apoptotic HGEC exposed to the biofilm using confocal microscopy at ×600 magnification. Positive control - complete TUNEL assay of DNAse-treated HGEC; negative control - TUNEL label solution only on HGEC in plain KSFM; unchalleged controls - complete TUNEL assay of HGEC in plain KSFM without biofilm challenge; biofilm on ring - HGEC challenged for 4 h and 24 h with the biofilm on a ring support (see Fig. 3A). (C) Percentage of TUNEL positive apoptotic HGEC exposed to the biofilm, estimated using confocal microscopy. Unchalleged controls - complete TUNEL assay of HGEC in plain KSFM without biofilm challenge; negative control - TUNEL label solution only on HGEC in plain KSFM; positive control - complete TUNEL assay of DNAse-treated HGEC; biofilm on ring - HGEC challenged with the biofilm on a ring support (see Fig. 3A) for 4 and 24 h. Values represent the mean ± SD of at least 4 fields of vision from 2 assays.